EPA-660/2-74-036
May 1974
Environmental Protection Technology Series
Development of Field Applied DDT
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
ft. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and .non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.
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EPA-660/2-74-036
May
DEVELOPMENT OF FIELD APPLIED DDT
By
K. H. Sweeny
J. R. Fischer
A. F. Graefe
H. L. Marcus
D. H. W. Liu
Contract 14-12-922
Program Element 1BB039
Project Officer
Dr. H. P. Nicholson
United States Environmental Protection Agency
Southeast Environmental Research Laboratory
College Station Road
Athens, Georgia 30601
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D. C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.45
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EPA Review Notice.
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or rec-
ommendation for use.
11
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ABSTRACT
Laboratory studies were carried out as a part of initial development
of a concept of controlled destruction of field applied DDT pesticide.
Copper-catalyzed aluminum reductant was shown to degrade DDT in
24 hrs at 25°C and 4 hrs at 40°C, without forming DDE. Copper-
catalyzed iron required a week to reduce DDT at 25°C and 8 hrs at
40°C.
Acidity for field degradation of DDT can be supplied by solid acids such
as sulfamic, oxalic, or citric. An integrated degradable particle was
demonstrated by a 5 /*m reductant particle overlaid with sulfamic acid
and coated with DDT. Only moisture is needed to initiate decomposi-
tion. In a demonstration, 98. 4% of the DDT was destroyed in 6 days
and 99. 8% in 2 weeks at 25°C.
Product TTTB is 50-fold less fat-soluble than DDT, and nearly insol-
uble in water. Product DDEt is 20-fold more soluble than DDT in
water. The vapor pressure of DDEt is about 80-fold greater than DDT.
Exposure of fathead minnows, bluegills, and rainbow trout to water
.saturated with DDEt {. 05 ppm) or TTTB (^001 ppm) produced no acute
toxic effects. The TLm of DDEt to Daphnia is about 35 ppb.
Long-term chronic exposure of fathead minnows to DDEt-saturated
waters showed no effect on adult growth and survival, egg production,
or hatchability. Growth and survival of freshly-hatched fry were
affected by DDEt above about . 006 ppm.
No effect on fathead minnow adult or fry growth and survival, egg pro-
duction, or hatchability was shown by TTTB-saturated water.
Nearly mature fathead minnows consumed 10 mg/kg body weight/day of
DDEt or 980 mg/kg body weight/day of TTTB in food without apparent
deleterious effect.
This report was submitted in fulfillment of Contract 14-12-922 under
the sponsorship of the Water Quality Office, Environmental Protection
Agency. '
See Glossary for chemical formulas and toxicological definitions.
111
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV DEVELOPMENT OF OPTIMAL DDT DEGRADATION 7
REACTION CONDITIONS
V CHARACTERIZATION OF DDT DEGRADATION 35
PRODUCTS
VI TOXICITY TESTING OF DDT DEGRADATION 47
PRODUCTS
VII ANALYSIS OF STUDIES 77
VIII ACKNOWLEDGEMENTS 83
IX REFERENCES 85
X PATENTS AND PUBLICATIONS 89
XI GLOSSARY OF PESTICIDES AND DEGRADATION
PRODUCTS 91
XII APPENDIX 95
IV
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FIGURES
No. Page
1. Dose-Response Curve for Daphnia magna
Exposed to Solutions of DDEt for 48 hours 58
2. Effect of Feeding High Dose Rate of DDEt
on Fathead Minnow 68
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TABLES
No. Page
1. Effect of Time on Al- Cu Reduction of DDT at 23-25°C 10
2. Effect of Time on Al- Cu Reduction of DDT at 40°C 11
3. Effect of Acid Type on Yield of TTTB from Al' Cu
Reduction of DDT 12
4. Reduction of DDT with Al- Cu Alloy 13
5. Effect of Time on Fe-Cu Reduction of DDT at 25°C 14
6. Effect of Time on Fe' Cu Reduction of DDT at 40°C 15
7. Effect of Acid Type on Reduction of DDT by Fe- Cu
Reductant 16
8. Effect of Amount of Zn* Cu Reductant on Reduction of DDT 17
9. Effect of Amount of Copper Catalyst on Reduction of
DDT by Zinc 18
10. Effect of Varying Zn/Cu Ratio on Extent of Reduction
of DDT 19
11. Efficacy of Zn-Cu Alloy for Reduction of DDT 20
12. Characteristics of Candidate Solid Acids 21
13. Efficacy of Selected Solid Acids in Reduction of DDT
by Zn» Cu Couple 23
14. Efficacy of Selected Solid Acids in Reduction of DDT
by Al- Cu Couple 24
15. Efficacy of Oxalic, Sulfamic and Citric Acids in
Al- Cu Alloy Reduction of DDT 25
16. Efficacy of Selected Acids in the Reduction of DDT
by Fe-Cu Couple 26
17. Analysis of Integral Zn- Cu Reductant - DDT - Acid
Particles After Reaction 27
VI
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TABLES
(Continued)
No. Page
18. Analyses of Integral DDT - Zn-Cu Citric Acid
Particles After Reaction 29
19. Analyses of Integral DDT Zn-Cu Citric Acid
Particles After Reaction 30
20. Zinc Consumed on Acid Coating and After Reaction
with DDT 31
21. Effect of Catalyst on Zinc Reduction of DDT 32
22. Effect of Catalyst on Aluminum Reduction of DDT 33
23. Effect of Catalyst on Iron Reduction of DDT 34
24. Solubility of DDEt in Water at 20°C 35
/ \
25. Solubility of DDT, DDEt and TTTB in Triolein 37
26. Solubility of TTTB in Various Solvents 38
27. Vapor Pressure of DDEt 39
28. Calculated Evaporation Rates of DDEt andDDT 39
29. Residue After Evaporation from Al' Cu Reduction of DDT 41
30. Infrared Spectra of Product from Al- Cu Reduction of DDT 42
31. Chemical Analysis of Raw Water Source for Fish
Biology Laboratory 50
32. Mean Weight and Length of Test Bluegill and Trout
Used in Acute Toxicity Testing 51
33. Mortality After 96 hours Exposure 53
34. Cumulative Percent Mortality of D. magna Exposed
to Different Concentrations of DDEt and DDT (Test 1) 55
35. Cumulative Percent Mortality of D. magna Exposed
to Different Concentrations of DD~E~t and DDT (Test 2) 55
36. Cumulative Percent Mortality of D. magna Exposed
to Different Concentrations of DDEt and DDT and
fed Yeast 56
VII
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TABLES
(Continued)
No. Page
37. Percent Mortality of Adult D. magna Exposed to
DDEt or DDT for 48 Hours 57
38. Reproduction Data for Fathead Minnows Exposed
to DDEt-Laden Water 61
39. Thirty-day Growth and Survival of Fathead Minnows
in Control and DDEt-Saturated Water 61
40. Thirty-day Growth and Survival of Control Larvae
Exposed to Various Concentrations of DDEt-Laden
Water (Test 1) 63
41. Thirty-day Growth and Survival of Control Larvae
Exposed to Various Concentrations of DDEt-Laden
Water (Test 2) 63
42. Thirty-day Growth and Survival of Fathead Minnow
Larvae Exposed to Diluted TTTB-Saturated Water 64
43. Acute Toxicity of DDEt to Fathead Minnows fed
DDEt-Laden Food for 10 Days 66
44. Mortality of Fathead Minnows Fed Different Dosages
of DDEt in Their Diet 69
45. Growth of Fathead Minnows Fed DDEt-Laden Food
for 28 Weeks 70
46. Egg Production and Hatchability by Fathead Minnows
Fed DDEt-Laden Food 71
47. Percent Survival and Mean Length of Fathead Minnow
Larvae Fed DDEt-Laden Food 72
48. Mortality of Fathead Minnows Fed Different Dosages
of TTTB in Their Diet 73
49. Growth of Fathead Minnows Fed TTTB-Laden Food
for 28 Weeks 74
50. Egg Production and Hatchability by Fathead Minnows
Fed TTTB-Laden Food 75
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TABLES
(Continued)
No.
51. Thirty-day Growth and Survival of Fathead Minnow
Larvae Fed TTTB-Laden Food ... 76
52. Theoretical Metal Usage and Reductant Cost for
Reductive Degradation of DDT 78
53. Calculated Effect of Particle Size on the Mean
Particle-Particle Distance for Al* Cu and Fe- Cu
Reductants, Integral Self-Destructing Pesticide
Concept 79
IX
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SECTION I
CONCLUSIONS
• Demonstration of the production and effectiveness of an
integrated self-destructing pesticide particle was provided when 5/U.m
copper-catalyzed zinc particles were overlaid with sulfamic acid and
the whole overlaid with DDT. Only moisture need be added for reaction,
and the addition of a slowly dissolving or permeating membrane between
the acid and DDT layer will give the desired delayed destruction. The
integral particle is of such size that it can be dispersed by spraying or
dusting. In one test, 98.4% of the DDT was destroyed in 6 days at
23-25°C, while in other tests an average of 99. 8% of the DDT was
destroyed in about 2 weeks at 23-25°C.
• Tests of the effect of time on the reduction of DDT by copper-
catalyzed aluminum at 23-25°C showed that the DDT was essentially com-
pletely consumed in 24 hrs (<0. 2-0. 3% DDT) with no DDE as a product.
Principal products were DDA and TTTB in nearly equal quantities. When
the temperature was raised to 40 C, essentially complete degradation
of DDT was obtained in 4 hrs, with the principal products being 2 1/2
3 parts TTTB to 1 part DDA. When iron catalyzed with copper was used
as a reductant, the DDT was slowly consumed over a week at 25 C, with
about 80% being destroyed in the first 24 hrs. The principal product
was TTTB, with DDA occurring only in trace quantities. Increasing the
reaction temperature to 40 C resulted in essentially complete reduction
in 8 hrs; again TTTB was the principal degradation product.
• An alloy of aluminum and copper (5. 4% Cu) was found to
give essentially the same results as a surface-deposited copper cata-
lyst in reducing DDT when a strong acid was used to provide the requi-
site acidity, although no reaction was shown with weak acetic acid. A
series of metal couples were examined in an effort to find an improved
catalyst for the A1-, Fe-. or Zn-catalyzed reduction of DDT. The
transition and neighboring group metals examined were not as effective
as copper in catalyzing the reduction of DDT.
• Practical field application of a self-destructing form of
DDT would require the use of a solid-form acid to supply the requisite
acidity in situ. Tests with copper-catalyzed aluminum reductant revealed
that oxalic, sulfamic, tartaric and citric acids were effective, while only
the oxalic and sulfamic acids were effective with aluminum-copper alloy
reductant. Copper-catalyzed iron reductant was effective with sulfamic
acid, while oxalic acid was intermediate in effectiveness. Zinc-copper
reduction of DDT could be achieved with either sulfamic or citric acids.
• A large excess of reductant, ranging up to 5 parts reductant/
part DDT or DDD was found effective in producing minimal DDD, but
was not effective in reductively degrading DDE.
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• Exposure of fathead minnows, bluegills, and rainbow trout
to water saturated with DDEt or TTTB (concentrations respectively
0. 05 and ^0. 001 ppm) resulted in no deaths or untoward behavior in
96-hr acute toxicity tests.
• Long-term chronic exposure of fathead minnows to waters
saturated with TTTB appears to have no deleterious effect on adult
growth and survival, egg production, egg hatchability, or the growth
and survival of freshly-hatched minnow fry.
• Long-term chronic toxicity tests with fathead minnows
showed that water saturated with DDEt had no deleterious effect on the
survival and growth rate, egg production, and egg hatchability. How-
ever, freshly-hatched minnow fry survived poorly in DDEt-saturated
water; the maximum concentration of DDEt which causes no effect on
the fry appears to be about 0. 006 ppm.
• Nearly mature fathead minnows can consume DDEt with
their diet at a rate of 10 mg per.-kilogram body weight per day and TTTB
at a rate of 980 mg per kilogram body weight per day without effect on
growth, survival, and egg production. These dosages may not have
any effect upon hatchability of eggs, but due to insufficient data it was
not possible to ascertain the effects of DDEt- or TTTB-laden food on
egg hatchability or larval growth and survival.
• DDEt, the principal product of zinc reduction of DDT, was
found to be soluble in water to the extent of about 50-70 ppb, or about
20-fold greater than DDT. The solubility of TTTB, the product of
catalyzed aluminum or iron degradation of DDT, however, was too low
to be measured—1 ppb or lower. TTTB was found to be about 50-fold
less soluble than DDT in .a fat (triolein), while DDEt was about 2-1/2
times more soluble than DDT in the same medium.
• The vapor pressure of DDEt was determined to be about
80-fold greater than DDT at 23-25°C. A calculation shows that the
rate of evaporative loss of DDT from an acre of glass would be one
pound per 830 days, while the DDEt produced from one pound of DDT
would last only 9 days.
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SECTION II
RECOMMENDATIONS
The practicality of the concept of controlled degradation of field-applied
DDT was believed amply demonstrated by the degradation of an integral
particle containing the reductant and requisite acidity, overlaid with
DDT - requiring only moisture to initiate decomposition. Further
application of this important concept to the control of the persistence
of chlorinated hydrocarbon pesticides is strongly recommended. Rec-
ommended activities include:
• Development of a controlled delay technique to permit pest
control action for a stated period prior to the initiation of degradation.
• Tests to assure the pest control effectiveness of the inte-
grated particle overlaid with DDT, and to demonstrate lack of phytotoxic
effects.
• Studies to ascertain the most effective means for dissemina-
tion of the controlled-degrading form of DDT.
• Small-scale field tests of the controlled-persistence DDT,
to establish the overall effectiveness, safety, cost, application hardware
requirements, crop compatibility, soil residual agents, and reductant
metal contamination implications.
• Modification of the concept for application to other field-
applied chlorinated hydrocarbon pesticides, such as toxaphene, or
heptachlor, including necessary toxic testing of degradation products.
Although the process leading to the formation of TTTB (Al» Cu or Fe» Cu
reduction) appears safe to the fish tested, the effect of these degradation
products on the shells of fish-eating or raptorial birds should be exam-
ined in suitable models in order to establish whether the "thin-shell"
syndrome is exhibited.
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SECTION HI
INTRODUCTION
The objective of this study has been the development of a system for
field-applied DDT which will degrade to a form harmless to life forms
after a suitable delay to allow for pest control action. Thus, the con-
cept is to provide a system having controlled persistence for chlori-
nated hydrocarbon type pesticides. The feasibility of the concept was
shown in earlier studies on Contract 14-12-596 (Reference 1).
Two basic areas were investigated in these studies. In the first, the
further development of the concept of degradation of a particle of
DDT under simulated field conditions was carried out. In the second,
the acute and long-term (chronic) toxicity of the two principal products
of the reductive degradation of DDT, DDEt, and TTTB, were tested
with appropriate fish species.
The earlier feasibility studies (Reference 1) had shown that DDT could
readily be reductively degraded by either of two basic methods. In
one, the reduction by zinc led to the formation of DDEt, in which all
three aliphatic chlorines have been removed. In the second, treatment
with aluminum produced a large molecule, TTTB, by reductively con-
densing two DDT molecules with the elimination of one atom of chlorine
per DDT. Both reductive reactions were found to be catalyzed by small
amounts of copper metal. In the studies to be described in this report,
'the reductive degradation of DDT has been examined in some detail.
Particular emphasis has been placed on the use of catalyzed aluminum
and catalyzed iron reductants, since the addition of these metals to the
soil would appear to offer little environmental problem. Excessive
zinc, on the other hand, could cause toxic problems to fish, although
no problems were believed attendant to the use of zinc reductant at the
levels employed in this study. The effect of continued metal application
from repeated applications would require additional analysis.
Important to the practical application of this concept of controlled deg-
radation of DDT is the development of a means for assuring a high
probability of the degradation reaction taking place at the designated
time. The achievement of reliable reaction conditions was believed to
be demonstrated by studies with an integrated particle, in which a
micron-sized catalyzed reductant particle was coated with a solid acid
to provide the requisite pH for reaction, and the DDT was overlaid on
the composite particle. Upon the addition of moisture, the reductive
reaction occurred. Increased probability of reaction because of the
close proximity of reactants, reduced reagent requirements in order
to insure complete reaction, and less possibility of phytotoxic damage
are some of the advantages of the integral particle concept. Since the
DDT is on the outside of the particle in unaltered form, the effective-
ness of the agent for pest control should not be diminished. This 5-10
•nicron particle would only require a time-delay membrane between
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the DDT layer and the solid acid-reductant particle to yield the desired
controlled persistence pesticide system. Particles of this size can be
readily sprayed with conventional equipment.
The development of the integral particle concept has required an eval-
uation of a variety of acids which might be used in practical systems.
A study of possible improved catalysts was also made.
A second phase of the effort involved the characterization of the prin-
cipal reaction products of reductive degradation of DDT. Product
studies included water solubility, vapor pressure of pure DDEt, the
hydrolytic stability, and resistance to further reduction.
The toxic testing to fish of the two principal degradation products of
reductive decomposition of DDT, DDEt and TTTB, consisted of the
examination of the acute toxicity to the fathead minnow and from this
study, to carry out long-term chronic studies on the same fish. The
long-term effect on survival, egg production, hatch rate, and survival
and growth of fry was examined. The acute toxicity to rainbow trout
and bluegills was also briefly studied. The acute and chronic bioassay
tests were performed using EPA recommended protocols.
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SECTION IV
DEVELOPMENT OF OPTIMAL DDT
DEGRADATION REACTION CONDITIONS
The study of the feasibility of controlled degradation of DDT carried
out under Contract 14-12-596 (Reference 1), disclosed two basic methods
for the reductive degradation of DDT. These techniques were selected
from a consideration of the following criteria:
• Degradation to proceed to the greatest extent possible,
removing a significant amount of chlorine from the
molecule or making the molecule biologically inert.
The products DDE and DDD were to be avoided if
possible. The major products should be harmless to
life forms, whether mammalian, fish, or bird life.
• The degradative reaction should proceed to a sub-
stantially complete destruction of DDT at ambient
temperature (^25 C) in periods of a week or less.
• The reaction should be capable of being carried out
in both soil and water, so that detoxification of both
of these media may be achieved.
• The degradative technique should be economically
feasible, and should not employ difficult to obtain
materials.
• The catalyst or degradative materials used should
not result in the introduction of harmful materials
into the environment.
In the first of these methods, catalyzed zinc reductant led to the removal
of the three aliphatic chlorines, leaving DDET as the principal product.
Zn-Cu,H
DDT DDEt
While this method proceeded smoothly to the stated product, some zinc
7
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ion was also produced. However, the analysis given in Reference 1
suggests that the zinc ion concentration will be so small so as not to
be a problem, unless repeated applications lead to excessive metal.
In the second technique, catalyzed aluminum, or possibly iron, can
be used as the reductant. In this case, a large, apparently insoluble
molecule is formed which is presumed to be biologically inert. This
species is formed by reductively couplirig two DDT molecules:
H-C C C — C-H
DDT TTTB
Since the presence of small quantities of aluminum or iron in runoff
waters would appear to offer no environmental hazard, this technique
was favored* Decreased reductant requirements also suggested that
the method might be more economical in operation, as well as pro-
viding less metal for absorption and build-up in the soil on repeated
applications.
ANALYTICAL TECHNIQUE
Principal analyses in this study were made using gas chromatography.
While the basic technique was described in Reference 1, some differ-
ences from the previous study were made in an effort to improve
accuracy and reliability. The analyses were made with a Perkin-
Elmer Model 990 Gas Chromatograph, equipped with dual flame ion-
ization and Ni 63 electron capture detectors; the flame ionization mode
was employed for most of the studies. Glass chromatographic columns
6 ft long and 4 mm ID packed with 2% SE-30 (methyl silicone gum rub-
ber) on silanized diatomaceous earth were used. Standard operating
conditions were nitrogen carrier gas at 80 cc/min, injection block
temperature 165 C, column temperature programmed from 140 to
240 C at 6 /min, and a detector manifold temperature of 220 C. The
sample volume injected was normally 1/>Q. Standardization curves
for all decomposition/reaction products were prepared and regularly
checked so that quantitative interpretation of the data could be achieved.
CATALYZED ALUMINUM REDUCTION
Reaction at 23-25°C
Earlier tests in which copper-catalyzed aluminum reductant was employed
for degrading DDT indicated that substantially complete reaction was
8
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achieved in 2 to 4 days at 25 C (Reference 1). However, the rate of
reaction had not been further defined. A series of tests was therefore
undertaken in an effort to define the time required to carry out this
reaction under both ordinary ambient conditions (/^ 25°C ) and simu-
lated summer weather temperature cycles. In the initial test, a reac-
tion was carried out for 25 hours at 25 C. In this test, 1 g of CP alum-
inum powder was added to 1 g of DDT in 20 ml of acetone, 1 meq of cop-
per ion was added to form the catalyst couple, and the solution was
acidified with 10 ml of 1. 5 N acetic or sulfuric acid. At the conclusion
of the test, the acetone-soluble products were filtered off for gas
chromatographic analysis. The remaining mass of unreacted aluminum,
copper catalyst, and TTTB was extracted with hot benzene to remove
the TTTB. The TTTB was collected and weighed. The TTTB collected
represented 33. 2% of the weight of DDT when acetic acid was used, and
37. 7% when sulfuric acid was employed. The benzene extract had a
melting point in each case of 270°G (uncorrected), which is the same
as that obtained previously with TTTB.
A second series was initiated in which the reaction was allowed to pro-
ceed for several different times at 25 C; acetic acid was used in these
tests. Initially, the results showed that when reaction had occurred
(i. e. , nearly all of the DDT was consumed), a balance of products
accounting for only about one half of the initial DDT was obtained.
Although the possibility that a product not responding to the gas chroma-
tograph, such as the 1,1, 4, 4-tetra(p-chlorophenyl)-2, 3-dichlorobutene-2
product (TTDB) identified by Mosier, Guenzi and Miller on photochemi-
cal decomposition of DDT (Reference 2), might account for the poor
balance, an additional product was believed present. Analysis of the
acetone-soluble fraction revealed the presence of DDA, which does not
respond to the gas chromatograph unless a derivative (e. g. , methyl or
silyl ester) is formed. Extraction, precipitation, and recrystallization
of the acetone extract revealed a substantial amount of DDA in the sam-
ples. The identity of the product was confirmed by melting point,
including mixed melting point tests with known material, and infrared
analyses. The results of tests after reaction for 8, 24, 48, 96, and
168 hrs ^action at 23-25°C are shown in Table 1.
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Analysis , %, after Reaction, hrs
8
0.2
1.3
2.1
1.3
68. 0
17.0
10.9
1.0
101.8
24
0.6
6.9
4.8
2.8
0.3
36.6
38.0
1.1
91.1
48
0.9
5.3
9.0
3.4
0.3
40.8
34.6
1.4
95.7
96
2.0
5.6
6.6
2.1
0.2
50.0
26.6
1.4
94.5
168
0.9
8.1
7.6
3.2
0.2
39.1
34. 0
1.5
94.6
TABLE 1
EFFECT OF TIME ON Al« Cu REDUCTION OF DDT* AT 23-25°C
Component
DDEt
DBF
DBH
ODD
DDT
TTTB
DDA
Minor Com-
ponents
Balance
1 g of DDT in 20 ml acetone, 1 g of Al powder catalyzed with 1 meq
Cu ion was added and the mix acidified with 10 ml 1. 5 N acetic acid.
Reactants were stirred with a magnetic stirrer.
#
Calculated as equivalent % DDT.
These results have been calculated on the basis of the equivalent per-
centage of DDT reacting, so that a material balance can be made.
The results show clearly that for 24 hrs reaction (or longer), the DDT
is nearly completely destroyed (0. 2-0. 3% remaining), with TTTB
and DDA as principal products. Since DDA is a principal product of
mammalian metabolism of DDT, it was considered a safe product.
DDE was not found and minimal amounts of DDD were shown. Moder-
ate amounts of DBP and DBH, which are also known metabolites of
DDT, were also determined (Reference 3).
The data shown in Table 1 have been examined by standard reaction
kinetic procedures. Although the test times provided practical data
on the time required to effectively decompose DDT, the time periods
were not satisfactory to determine the reaction rate for the disappear-
ance of DDT. An attempt was made to determine the rea'ction order for
the appearance of TTTB and DDA. Although the data for TTTB appear-
ance fit a 1st order plot reasonably well at 24, 48 and 96 hrs, the 8-hr
and 168-hr points deviate significantly. The rate of appearance of DDA
likewise can be reasonably represented by 1st or 2nd order kinetics
10
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after 24, 48 or 96 hrs, but the initial (8-hr) and final (168-hr) points
deviate sharply. The existence of several minor products, as well as
the two major products, suggests that either parallel or consecutive
reactions are probably being obtained so that a clear-cut kinetic analy-
sis of the data would be difficult, and outside the scope of these studies.
Reaction at 40°C
The extent of reaction after 1, 2, 4, 8, and 24 hrs reaction at 40°C was
also determined. This test is a simulation of conditions which might
exist in the summer in southern agricultural lands. Temperatures as
high as 45 C are reported for Georgia soils (Reference 4). These
results follow in Table 2.
TABLE 2
EFFECT OF TIME ON AL Cu REDUCTION OF DDT* AT 40°C
Component
DDEt
DBF
DBH
DDD
DDT
TTTB
DDA
Minor Com-
ponents
Balance
Analysis , %, after Reaction, hrs
1
0.1
1.3
-
0.9
72.0
14.3
8.2
1.0
97.9
2
0.3
1.6
0.4
1.6
62.0
18.9
11.6
1.2
97.6
4
1.3
4.0
1.5
4.3
0.6
58.3
19.7
6.7
96.4
8
1.6
5.4
5.2
4.2
0.7
54.3
21.0
2.4
94.8
24
1.7
5.6
6.1
3.9
0.9
55. 5
19.7
2.4
95.8
Conditions, except for temperature, same as in Table 1,
#
Calculated as equivalent % DDT.
These data indicate that substantially all of the DDT has reacted after
4 hrs at 40°, comparable to 24 hrs at 25 C. Again, the sum of the
TTTB and DDA is approximately 75% after 4ohrs or more reaction, but
the product is predominantly TTTB at the 40 C reaction temperature.
The data in Table 2 could not be represented by any simple kinetic rate
equation. Both the formation of TTTB and of DDA were examined.
11
-------
Use of Sulfuric or Acetic Acids^
There appears to be a further factor involved in the reaction related
to the acid employed. In one set of experiments, the reaction of DDT
with Al» Cu reductant was carried out in parallel using both acetic and
sulfuric acids as the acidifiers. In these tests, 1 g of DDT in 20 ml of
acetone was treated with 1 g of aluminums, powder catalyzed by the addi-
tion of 1 meq of copper. The acidity was provided by adding 10 ml of
1. 5 N acid. The weight of TTTB and DDA, after 24 and 141 hrs reac-
tion at 25°C is shown in Table 3. »
TABLE 3
EFFECT OF ACID TYPE ON YIELD OF TTTB
FROM Al- Cu REDUCTION OF DDT
TTTB*, % PDA? %
24 hr Reaction
Sulfuric Acid 59. 8 15. 5
Acetic Acid 32. 4 33. 6
141 hr Reaction
Sulfuric Acid 48.2 10.2
Acetic Acid 42.1 2 7. 7
* «-
Calculated as equivalent % DDT.
These results show that the amount of TTTB produced was greater
when sulfuric acid was employed than for those runs in which the
acidity was provided by acetic acid, and that more DDA was obtained
when acetic acid was used. The effect of acid will be explored fur-
ther in another section of this report.
Reduction with Al-Cu Alloy
It would be important to determine whether a finely divided aluminum-
copper alloy powder could be used in place of the freshly-prepared
couple currently employed as a reductant, since the alloy would repre-
sent a practical means for using copper-catalyzed aluminum reductant.
A sample of an aluminum-copper alloy powder containing 5.4% copper
(particle size: 90% passing 44-micron sieve) was supplied for this
analysis by Reynolds Metals Company.
Two determinations were made. In each, 1 g of alloy was reacted with
1 g of DDT in 20 ml of acetone. However, in one sample the acidity
12
-------
was supplied by 10 ml of 1. 5 N sulfuric acid; in the other 10 ml of 1. 5
acetic acid. In both cases the reaction was carried out for 24 hrs at
ambient temperature (23-25°C). The results of TTTB and DDA pre-
cipitation, and gas chromatography of the ace tone-soluble fraction,
follow i n Table 4.
TABLE 4
REDUCTION OF DDT WITH Al- Cu ALLOY*
Analysis , after Reaction for 24 hrs at 23-25°C
Component Sulfuric Acid Acetic Acid
TTTB 72. 7 0. 6
DDA 6. 6
DDEt 2..8 <0. 1
DBF 1. 3 1.0
DDMU - 0.2
DDMS - <0. 1
? 1. 3 0.2
DDD 1.0 0.4
DDT 0.4 100. 0
Balance 86.1 102.4
*
1 g of DDT in 20 ml acetone, 1 g Al* Cu powder, and acidified with
10 ml of 1. 5 N acid. Reactants stirred with magnetic stirrer.
$$
Calculated as equivalent % DDT.
A clear difference is shown between these samples. The reaction car-
ried out in an acetic acid medium has clearly not proceeded to a useful
state. However, when sulfuric acid was employed,: the reaction pro-
ceeded largely to TTTB (72. 7% of DDT converted to TTTB). The result
with sulfuric acid is comparable to that obtained when a freshly-formed
Al» Cu couple is employed.
CATALYZED IRON REDUCTION
Reaction at 23-25°C
It was of interest to examine the rate and extent of reaction when DDT
was reduced by catalyzed iron reductant. Although some data had been
reported for isolated tests of this system, a series to show the extent
13
-------
of reaction with reaction time had not been carried out. In this series,
1 g of DDT was reacted with 1 g of powdered iron to which 1 meq of cop-
per ion had been added to form the couple. The solution (2:1 acetone-
water) was O. 5 N in acetic acid. The results of analyses on these sam-
ples, expressed as equivalent DDT percent, are given in Table 5.
TABLE 5
Analysis , %, after Reaction, hrs
24
1.1
0.9
1.7
12.9
16. 7
59. 6
92.9
48
2.4
1. 7
2. 0
15.5
9.9
64.3
95.8
96
1.3
0.8
1.9
15.1
7.2
63.5
89.8
168
1.2
0.8
1.9
14.3
2.4
69.3
89.9
-jfr.
EFFECT OF TIME ON Fe-Cu REDUCTION OF DDT AT 25°C
Component
DDEt
DBF
DDE
ODD
DDT
TTTB
B alance
*
1 g of DDT in 20 ml acetone, 1 g of CP powdered iron catalyzed
with copper ion was added and the mix was acidified with 10 ml
of 1. 5 N acetic acid. The reactants were stirred with a blade
stirrer.
##
Expressed as equivalent % DDT.
These results indicate a first order consumption of DDT with TTTB as
the principal product. The iron system does not appear to be as rapid
as the copper-catalyzed aluminum or zinc systems. Only trace quan-
tities of DDA were found when copper-catalyzed iron reductant was
used, in contrast to the results with the Al»Cu system in which TTTB
and DDA were found in nearly equal quantities.
Reaction at 40°C
The effect of increasing the reaction temperature to 40 C, simulating
extreme summer soil conditions, was also noted. The results of
analyses follow in Table 6.
14
-------
TABLE 6
EFFECT OF TIME ON Fe« Cu REDUCTION* OF DDT AT 40°C
_ Analysis , %, after Reaction, hrs
Component
DDEt
DDD
DDT
TTTB
Minor Com-
ponents (DBF,
DDMS, DDMU,
etc. ) 2.0 1. 8 2.1 1. 8 2.9
4
0.9
10.4
9.5
77. 7
8
1. 0
10.1
0.4
93.9
17
1.5
7. 3
0.4
93. 7
24
1.2
14.9
0.0
88.4
72
4.8
3.1
0.2
78. 0
Balance 100.5 107.2 105.0 106.3 89.0
Conditions, except temperature, same as Table 5.
$$
Converted to equivalent % DDT from which component was produced.
These results indicate that the increase in temperature from 25 to
40 C produced a striking increase in the rate of reaction, since essen-
tially complete reaction was obtained in 8 hrs at 40 while 168 hrs at
25 was required for an equivalent reaction. It should be noted that the
copper-catalyzed iron reduction of DDT leads to nearly complete con-
version of DDT to the insoluble product TTTB; however, it appears that
the TTTB may be partially consumed at reaction periods greater than
17 hrs. The balance after 72 hrs reaction may indicate the formation
of a new product which does not respond to the gas chromatograph. An
attempt to fit the data to kinetic rate equations was unsuccessful.
Use of Sulfuric or Sulfamic Acids
Following tests with the Al« Cu system described later in this section
on the effect of acid type on the reaction, tests were carried out in
which sulfuric and sulfamic acids were used for acidifying the reactants.
The reaction was carried out in the same manner as described above.
In either case the reaction mix was 0. 5 N in acid. The results of tests
are presented in Table 7.
15
-------
• Sulfuric
0. 7
1.8
, L6
3.4
64.5
10.5
82. 5
Sulfamic
2.8
3.3
1. 7
15.1
0.1
64. 7
si 7
TABLE 7
EFFECT OF ACID TYPE ON REDUCTION
OF DDT BY Fe» Cu REDUCTANT*
## o
Analysis , %, after 24 hr Reaction at 25 C
Component Acid:
DDEt
DBF
DDE
DDD
DDT
TTTB
Balance
1 g of DDT in 20 ml acetone and 1 g of CP powdered iron catalyzed
with 1 meq of copper ion was added and the mix was acidified with
10 ml of 1. 5 N acid. The reactants were stirred with a blade stirrer.
##
Expressed as equivalent % DDT.
It is interesting to note that while a large portion of DDT was unreacted
when sulfuric acid was employed, essentially complete consumption of
the DDT was achieved with sulfamic acid, producing largely TTTB as
a product. Negligible amounts of DDA were found.
CATALYZED ZINC REDUCTION
Consideration of the catalyzed zinc reductant system would require the
use of minimum amounts of zinc, since excessive zinc ion is toxic to
fish (Reference 1). A limited series of experiments was carried out
in an effort to establish minimum zinc-to-DDT ratio for effective reduc-
tion. The previous tests (Reference 1} had employed equal weights of
zinc and DDT (the theoretical zinc required to reduce 1 g of DDT to
DDEt is 0.276 g) or a 2-1/2-fold excess over stoichiometry.
In these tests, from 0.1 to 3 g of Zn per g of DDT was used; the ratio
of copper catalyzt to zinc was held constant at 1 meq copper ion added
per g Zn.
In the new series, 20 ml of acetone containing 1 g DDT was. added to the
specified amount of zinc, the given amount of copper ion was added to
form the Zn« Cu couple, and the solution was made 0. 50 N in acetic acid.
The reactions were carried out for 2 hrs at ambient temperature (23-
25 C). The results of analyses are shown in Table 8.
16
-------
5.4
0.5
9.6
77.4
12.2
1.4
22.5
48.8
21.6
1. 5
41.1
26.9
29.5
2.3
43.0
5.0
34.8
2.5
45.5
2.8
TABLE 8
EFFECT OF AMOUNT OF Zn« Cu REDUCTANT
ON REDUCTION OF DDT*
Analyses , %, after 2 hr Reaction at 25°C
Component g Zn/g DDT: 0.10 0.20 TQTJTJT^Q" TJTgTJ
DDEt
DDMS
DDD
DDT
Minor Com-
ponents (DBF,
DMC, DDMU,
etc. )
B alance
Component g Zn/g DDT:
DDEt
DDMS
DDD
DDT
Minor Com-
ponents (DBF,
DMC, DDMU,
etc.)
Balance
*
1 g of DDT added in 20 ml acetone, followed by given amounts of CP
zinc dust. Copper ion was added at the rate of 1 meq/g Zn dust and
the mix was acidified with 10 ml 1. 5 N acetic acid. The reactants
were stirred with a magnetic stirrer.
Converted to equivalent % DDT.
These results show a dramatic decrease in the DDD obtained, with an
equivalent increase in DDEt production, as the ratio of zinc to DDT is
increased. These results show that a large excess of reagent will force
the reaction to the desired products. Indeed, it suggests that a reactor
configuration in which a large excess of reagent is present (such as a'
packed bed) may be the preferred method of reaction for such applications
17
1.1
98.2
0. 75
41.8
4.8
40.2
1.1
3.2
90.9
1.0
52.7
7.1
30.0
_
3.5
95.3
1.5
61.4
6.2
21.8
_
4.1
84.3
2.0
71.2
6.2
8.1
_
4.2
90.4
3.0
76.6
7.8
3.5
—
4.6
92.3
4.6
94.9
4.9
94.3
4.4
89.9
3.6
MW^HB^M
91.7
-------
0.25
37.2
8.7
32.5
0.50
45. 7
6.0
33.1
1. 0
42.3
5.6
34.3
1.5
48. 0
3.7
31.6
2. 0
55.1
7.2
23.0
5. 0
38.1
5.2
38.8
as industrial waste treatment.
In an additional series of tests, the effect of changing the ratio of cop-
per catalyst to zinc reductant was studied. These tests were carried
out in the same manner as the previous series except that 1 g of zinc
was used per g of DDT, and the amount of added copper ion was varied.
These tests were also carried out for 2 hrs at ambient temperatures
(25°C). The results follow in Table 9.
TABLE 9
EFFECT OF AMOUNT OF COPPER CATALYST ON
REDUCTION OF DDT BY ZINC*
Analysis , %, after 2 hr Reaction at 25°C
Component Cu, meq: 0. 25
DDEt
DDMS
ODD
DDT
Minor Com-
ponents (DDMU,
DBH, etc. )
Balance
Procedure same as used in Table 8, except that 1 g Zn dust was
used and added copper ion was varied per table.
Converted to equivalent % DDT.
These results do not show an identifiable effect on the reaction as the
amount of catalyst is changed, except that the reaction at the extremes
of catalyst (0. 25 and 5. 0 meq Cu/g Zn) appear to give a poorer yield
of DDEt. The best DDEt production (and lowest DDD) was given at
2. 0 meq Cu/g Zn.
In another test, a 1 g sample of DDT was reacted with 3 g of zinc dust
to which 1 meq of copper ion was added. The data from this test indi-
cates less conversion than was obtained when 3 meq of Cu was used
with 3 g of zinc reductant.
5.0
85. 0
4.4
89.2
4.8
87. 0
5.0
88.3
4.8
90.1
4.2
86.3
18
-------
TABLE 10
EFFECT OF VARYING Zn/Cu RATIO ON
EXTENT OF REDUCTION OF DDT*
$$ Q
Analysis , %, after 2 hr Reaction at 25 C
Component 3 g Zn, 1 meq Cu 3 g Zn, 3 meq Cu*»*
DDEt 66.5 76.6
DDMS 11.2 7. 8
DDD 12. 1 3. 5
DDT
Minor Com-
ponents (DBF,
DDMU, DMC) 5. 9 3. 6
Balance 95. 7 91. 7
Procedure same as used in Table 8 and 9.
## ~.
Converted to equivalent % DDT.
Repeated from Table 9 for comparison.
Zinc-Copper Alloy Reductant
The zinc-copper reductant is normally prepared by adding copper ion
to slurried zinc powder, whereupon the copper ion precipitates onto
the surface of the zinc powder by electrochemical replacement. It
was of interest to determine whether similar efficacy of reduction could
be achieved if a zinc-copper alloy were used, since the use of an atom-
ized alloy powder might represent a practical means for preparation of
the catalyzed zinc. The optimal ratio of surf ace-deposited copper
appears to be about 1 meq/g zinc, which represents about 3% copper on
a total particle basis. Samples were prepared by blending zinc dust
and copper powder (3, 5, and 10%), placing the mix in a porcelain cru-
cible, and heating the mixture in an electric furnace while flooding the
furnace with nitrogen gas so as to reduce oxidation of the zinc. However,
despite these precautions, some ZnO appeared to have been formed.
The samples were heated to 875°C, well above the reported melting
point of the alloys (Reference 5) of about 460 for the 3% alloy, 500 for
the 5% composition, and 580 for the alloy containing 10% copper. The
alloys were then cooled, and the pellets were reduced to a. fine powder
by filing. Iron from the file was removed magnetically. The samples
were then reacted with DDT by the usual method: 1 g reductant was
reacted with stirring in 20 ml acetone + 10 ml 1. 5 N acetic acid with
1 g of DDT. The reaction was carried out for 26 hours at 25 C. The
19
-------
results are shown in Table 11.
TABLE 11
EFFICACY OF Zn-Cu ALLOY FOR REDUCTION OF DDT
Analysis , %, after 26 hr Reaction at 25°C
Com-
ponent
DDEt
DDMU
DDMS
DDD
DDT
Sample:
3% Cu
Alloy
57.4
2.8
10.0
29.7
5% Cu
Alloy
26.4
2.1
7.8
63.7
10% Cu
Alloy
48.0
3.3
11.3
37.4
Zn« Cu
Couple
82.1
3.8
12. 0
2.1
Zn
Dust
59.5
5.9
20. 7
13.8
Balance 99.9 100.0 100.0 100.0 99.9
#
Converted to equivalent % DDT.
While all of the alloy samples were effective in destroying DDT, none
was as effective as the freshly-deposited couple in forcing the reaction
to the desired product DDEt while forming minimal amounts of DDD.
It is of interest that the alloy sample containing the 3% copper appeared
to be the best of the alloys, although it was not quite as good in this
series as the uncatalyzed zinc.
EFFECT OF ACID TYPE ON DDT REDUCTION
The practical development of a controlled self-destructing form of DDT
for use in the field requires the combination of reductant, requisite
acid for the reaction, and a means for suitably delaying the degradation
process. A careful consideration of the requirements suggests further
that it would be desirable to employ a solid form of the acid since (1)
dissemination would be simpler, (2) minimum acid would be required
since it would be necessary to provide it only at the reaction site, and
(3) possible phytotoxic effects from the acid could be avoided. It has
been shown in earlier studies that reduction can be achieved by the
Zn* Cu couple in the presence of acetic, sulfuric or hydrochloric acids,
with the reaction proceeding best at a pH <4 (Reference 1). It remained,
to be established if other acids could be employed, and whether proper-
ties such as the solubility, acid strength (dissociation constant), etc. ,
would have an effect on the extent of the degradation reaction. A series
of eight solid state acids were examined and compared with acetic acid.
The acids and some of their chemical and physical properties are shown
in Table 12.
20
-------
TABLE-12,
CHARACTERISTICS OF CAN^I^ATE SOLID ACIDS
Acid
Sulfamic
Citric
Oxalic
Fumaric
Maleic
Anhydride
Suberic
Tartaric
Lactic
Acetic
Formula
H,N SO, OH
C* Lf
(COOH)CHxC(OH)-
(COOH)CH2COOH
(COOHJ2
(CH2COOH}2
(CHCOKO
w
(CH2)6(COOH)2
(CHOHCOOH)2
CH3CHOHCOOH
CH3COOH
v-*OS t
$/lb
0.15
0.30
0.21
0. 175
0. 14
-
0.415
0.275
0.09
Dissociation Constant
K K K
»-1 *2 ^3
7. 9xlO-1
8xlO"4 1. 8xlO"5 4x10
3.8xlO"2 4. 9xlO"5
IxlO"3 3xlO~5
1.5xlO"2 1.3xlO"6
S.OxlO"5
9. 6xlO"4 2.9xlO~5
1.4xlO"4
1.8xlO"5
Solubility
g/100 g
H20at
A ••• |V^^ ATI t~
j\ii. iDi cut
Temperature
14.7
13.3
10. 0
0.7
16. 3
0. 14
120
OO
oo
Not a solid acid; shown for comparison.
-------
These acids were selected on the basis of the solubility range and dis-
sociation constant, and all are common items of commerce.
The tests were made by adding 1 g of DDT in 20 ml acetone to a flask
containing 1 g of zinc powder. The copper couple was then formed by
adding with stirring 1 meq of copper ion in aqueous solution. To this
suspension, 10 ml of water was added, and 15 millimoles of the acid
was then added at the inception of the reaction. After two hours reac-
tion at ambient temperature (23-25 C), the reactants were filtered and
the solutions analyzed. The solutions were not analyzed for TTTB or
DDA. The results of these analyses are presented in Table 13.
An examination of these results shown that effective reduction was
achieved when sulfamic, citric, or acetic acid was used, and that
little reduction was achieved when maleic anhydride was employed as
the acid. Reduction was also achieved when the acidity was provided
by oxalic, fumaric, or suberic acids, although the reaction did not
proceed as far as when citric, acetic, or sulfamic acids were employed
(lower DDEt, higher DDD percentage when oxalic acid, etc. , used). In
examining the basis for the observed efficacy of action of these acids,
it may be noted that two of the less efficient acids, suberic and fumaric
acids, are only sparingly soluble. The efficient acids show a range of
dissociation constants. Sulfamic acid was the strongest acid employed,
acetic the weakest, and citric acid was intermediate in acidity. The
lack of product balance may indicate the formation of TTTB or DDA,
for which no analysis was made.
Similar- tests were carried out with the copper-catalyzed aluminum
reduction of DDT. In these tests, 1 g of DDT in 20 ml acetone was
added to a flask containing 1 g Al powder. The Cu couple was formed
by adding, with stirring, 1 meq of aqueous Cu ion. To this suspension,
10 ml of water and 15 millimoles of the acid were added. The suspen-
sion was reacted for 24 hrs at ambient temperature (23-25 C), filtered
and analyzed. The results of these analyses are presented in Table 14.
It is clear that most of the acids have given essentially complete reduc-
tion of the DDT. Only the suberic acid led to significant unreacted DDT.
The small amount of DDT (0. 3 - 0. 7%) in the remaining flasks is believed
to represent material splashed onto the flask wall where it was removed
from the reaction. The greatest yield of the product TTTB was shown
with oxalic, sulfamic, tartaric, and citric acids. The balance of pro-
ducts is poor and may represent at least in part DDA which was not ana-
lyzed for. These tests were carried out before DDA product was dis-
covered. The procedure used was believed adequate to recover DDT
and related products possibly trapped in the solids.
Since the alloy of aluminum and copper would appear to be the most
practical way of carrying out a copper-catalyzed aluminum reduction of
DDT in the field, a test of the efficacy of selected solid acids with this
system was also desirable. Sulfamic, citric, and oxalic acids were
examined, based on the above results. In these tests, 1 g of the Al* Cu
22
-------
TABLE 13
INJ
00
Com-
ponenl
DDEt
DBF
DDMS
DDD
DDT
B alance
EFFICACY OF SELECTED SOLID ACIDS IN REDUCTION*
OF DDT BY Zn- Cu COUPLE
. ##* „.
Analysis , %,
Oxalic
33. 1
0.3
4.2
42.8
-
80.4
Maleic
0.4
-
0.4
2.2
91.3
94.3
Fumaric
22.7
0.6
3.2
56.0
-
82.5
after 2 hr
Citric
52.4
0.8
5.2
29.1
-
87.5
Reaction at
Suberic
27.4
0.7
4.5
38.1
0.8
71.5
25°C
Sulfamic
54.6
2. 1
4.0
21.2
-
81.9
Acetic*
50.4
1.7
6.2
26.0
-
84. 3
##
*#*.
1 g of DDT dissolved in 20 ml acetone and 1 g Zn catalyzed with 1 meq
of Cu ion added. 10 ml of water added followed by 15 millimoles of
solid acid. Mixture stirred with magnetic stirrer.
Minor components not listed.
Converted to equivalent % DDT
+ Not a solid acid; shown for comparison.
No analysis made for TTTB and DDA.
-------
TABLE 14
EFFICACY OF SELECTED SOLID ACIDS IN
REDUCTION OF DDT BY Al« Cu COUPLE*
**
Com-
ponent
DDEt
DBP
DDE
DDD
DDT
TTTB
Balance
fi
Analysis, %, after 24 hr Reaction at 25 C
Acid: Lactic
47.5
Tartaric
Sulfamic
51.0
60.8
Citric
1.3
3.8
3.4
1.3
3.8
0.3
33.6
2.8
1. 1
-
1.3
4. 1
0.3
41.4
4.2
3.4
-
0.9
2.4
0. 1
49.8
2.4
1.6
-
1.2
3.8
0.3
39.8
49. 1
Maleic
Oxalic
Fumaric
B alance
51.4
69.7
42. 1
Suberic
DDEt
DPB
?
DDE
DDD
DDT
TTTB
1. 1
14. 1
-
1.4
2.4
0.7
31.7
3.2
1.4
-
2.2
4.4
1.6
56.9
2.3
2. 6
2.8
1. 1
4. 1
0.7
28.5
0.8
4.5
5.6
0.9
3.2
19.2
16.6
50.8
**
**#
jc
Procedure same as cited in Table 13 except aluminum
powder rather than zinc dust used as reductant.
Minor components (ca 1-2%) not listed.
Calculated as equivalent DDT reacted.
24
-------
alloy powder (particle size: 90% passing 44-/tm sieve, 5. 4 wt % cop
per), 1 g DDT, 20 ml acetone and 15 millimoles of the acid in 10 ml
water were reacted for 24 hr at ambient temperature (25°C). The
results of analyses of these samples follow:
TABLE 15
Analysis , %,
Oxalic
1.8
2.8
0.6
73. 7
not determined
after 24 hr Reaction
Sulfamic
1.6
3.3
0.5
69.2
4.9
at 25 C
Citric
0. 0
0.4
97. 0
0. 6
EFFICACY OF OXALIC, SULFAMIC AND CITRIC ACIDS
IN Al- Cu ALLOY REDUCTION OF DDT
Component
DDEt
ODD
DDT
TTTB
DDA
Minor Com-
ponents (DBP,
DBMS, DDMU,
etc.) 3.6 6.3 0.1
Balance - 85.8 98.1
Procedure same as cited in Tables 13 and 14 except that
1 g of Al* Cu alloy used instead of metal powder + copper
ion catalyst.
** nf
Converted to equivalent % DDT from which component was
produced.
In this system, oxalic acid appears to be about as effective as sulfamic
acid in degrading DDT, while the weaker, citric acid failed to degrade
the DDT. Strong acids are apparently required to promote the reduction
of DDT by Al* Cu alloy.
The effect of acid type on the reduction of DDT by copper -catalyzed iron
was also considered. In this series of tests, 1 g of powdered iron was
added to 1 g of DDT in 20 ml of acetone, and 1 meq of copper ion was
added to form the catalyst couple. The solution was then acidified by
adding 15 meq of the given acid in 10 ml of water. The results follow;
a previous result in which aqueous acetic acid was used as the acid
source is also given for comparison.
25
-------
TABLE 16
EFFICACY OF SELECTED ACIDS IN THE REDUCTION
OF DDT BY Fe' Cu COUPLE*
•{{5J5
Analysis , %, after 24 hr Reaction at 40 C
Component Acid: Sulfamic Citric Oxalic Acetic
DDEt 2.8 - 0.6 1.2
DDD 14.8 0.8 3.6 14. 9
DDT 0. 0 99. 0 60. 5 0. 0
TTTB 70.5 - 25.2 88.4
Minor Com-
ponents (DBP,
DDMS, DDMU) 2.7 0.2 1.8 1.8
Balance 90.8 100.0 91.7 106.3
*
Procedure consisted of dissolving 1 g of DDT in 20 ml acetone, adding 1
CP powdered iron powder and 1 meq copper ion catalyst, and then 10
ml of water. 15 millimoles of the acid was then added and the mixture
was stirred with a blade stirrer.
**
Converted to equivalent % DDT from which product was produced.
These results show that the sulfamic acid led to the effective reduction
of DDT, while weaker citric acid was not effective in converting DDT
to a less toxic form. Oxalic acid, which is nearly as strong as sulfamic
acid, led to intermediate results. The comparative dissociation constants
for the acids employed were shown in Table 12.
DEGRADATION OF INTEGRAL REDUCTANT-DDT-ACID PARTICLES
It has been shown that the best opportunity for degradation of DDT in
the field should be achieved in a system whereby the reductant, DDT,
and acid are kept in close proximity. An integral particle concept was
outlined in an earlier report in which the reductant particle, coated with
a solid acid and overlaid with the DDT, was considered to be a suitable
means for carrying out the degradation reaction in the field. If this
system, which could presumably be spray-applied or dusted, is shown
practical, then the controlled delay could be introduced by applying a
slowly-removed membrane between the solid acid-reductant and the
DDT. Since the DDT surface is exposed, the pest-control action should
be unaffected.
Tests of this concept were initiated and the results suggested that the
tests were successful. Zn« Cu couple was prepared by placing 5 g of
26
-------
5 ,u-m powdered Zn in a flask and adding 20 ml acetone, followed by 5
ml of 1 N CuCl2 solution to form the couple. The reactants were mixed
for 5 min, filtered, washed with acetone and air dried.
In the initial series, one gram samples of the Zn» Cu couple were
weighed into 50 ml beakers, and 10 ml of water was added. Then 5
millimoles of the solid acid (sulfamic acid or citric acid) was added
and the water was evaporated from the samples. In some designated
samples, 1 drop (~'0. 02 ml) of Triton X-100 surfactant was added to
aid dispersal. Some bubbling indicating a slight to moderate attack of
the acid on zinc was noted. In an additional test, ethanol was used as
the solvent for the acid; in this case the acid did not appear to attack
the metal.
After the samples had dried, 0. 25 g DDT in 5 ml acetone was added and
the acetone was evaporated. The samples were then placed on a 7 cm
filter paper in a petri dish, a drop of water was added, and the dish
covered. A drop of water was added daily to keep the paper moist.
After 6 days, at ambient temperature, the samples were washed with
acetone and analyzed. The results from some tests follow:
TABLE 17
ANALYSIS OF INTEGRAL Zn- Cu REDUCTANT-DDT-ACID
PARTICLES AFTER REACTION
*
Analysis of Residue , %, after
6 days at 24-26°C
Reductant: Zn* Cu Zn'Cu Zn« Cu
Com- Acid,: Sulfamic Citric ^ Citric
ponent Triton X-100: Ca 0. 02 ml Ca 0. 02 ml
DDEt 75. 6 43. 9 0. 7
DDMU 0.8
DDMS 16. 3 9. 1
DDE - - tr
DDD 5.7 28.0 1.2
DDT 1.6 15.8 98.1
Sample Recovered, % 49 53 57
&
All values converted to equivalent % DDT reacted.
$$
Ethanol solvent used for particle preparation.
27
-------
The samples employing citric acid with water as a solvent had a sticky
consistency and extensive reaction was not shown. However, the sam-
ple which had been acidified with sulfamic acid was a crumbly mass
which could be readily separated into discrete particles. It is impor-
tant to note that the DDT in this sample was nearly completely destroyed,
the residue assaying 1. 6% DDT. The residue was largely DDEt; the
DDEt and DDMS fractions accounted for about 92% of the residue. Sub-
stantial decomposition of the sample acidified with citric acid and using
ethanol as a solvent was also shown.
Quantitative recovery of the samples was not achieved, as noted by the
values given for the percent of sample recovered. While handling dif-
ficulties, especially with the very sticky citric acid samples, may con-
tribute to this problem, the unrecovered 50% in the sulfamic acid sam-
ple is believed due to other causes. The high evaporative loss of DDEt
may be the reason for the apparently poor recovery.
Based upon these results, a second series of tests was carried out.
A quantity of Zn« Cu couple was prepared and coated with either citric
acid or sulfamic acid, and tests were carried out on aliquot portions of
these batches.
The citric acid-coated particles were prepared by first slurrying 11 g
of zinc dust in acetone, adding the requisite copper ion to form the
couple, filtering and washing the couple, and drying under vacuum.
Citric acid monohydrate (55 millimoles, 11. 56 g) was then added to the
flask, and 25 ml absolute ethanol was added to dissolve the citric acid.
The solvent was then removed with difficulty in a rotary evaporator.
The mix tended to be viscous and frothy, although no bubbling indicative
of zinc reaction was noted. A weight loss equal to 9. 4% (theoretical,
8. 6%) of the citric acid monohydrate weight indicated that the acid was
dehydrated in the vacuum drying process. The mass was then ground
and mixed in an agate mortar, and 2. 5 g DDT in an acetone solution was
added, and the acetone was removed in the rotary evaporator. The sam-
ple was gently pulverized and split into 10 portions for testing. These
samples were placed onto 11 cm filter papers in petri dishes, spread
into a thin layer and moistened with a drop of water daily. A portion
of the sample was withdrawn periodically, extracted five times with
warm benzene and the solution analyzed by gas chromatography. The
analyses of some of the samples are shown in Table 18.
28
-------
TABLE 18
ANALYSES OF INTEGRAL DDT-Zn. Cu-CITRIC ACID
PARTICLES AFTER REACTION
Analysis, %, after time, hr, at 23-25 C
Component 0 20 70
DDEt 35.7 62.4 82.2
DDMU 2. 6 2. 8 2. 9
DDMS 6.2 9. 4 12. 7
DDD 7. 0 4.2 1.3
DDT 48.4 21.3 0.8
*
Analyses calculated as equivalent amount of DDT reacted; analyses
normalized to 100% because of differences in sample size.
An almost immediate reaction was noted and the zero time reaction
shown was believed due to reaction during the handling of the samples
following DDT application. It should be noted that DDD is being con-
sumed during the reaction. The favorable conversion of the DDT
using citric acid as the acidifier is somewhat surprising in view of
the results of Table 17 and evidently represents an improved prepa-
ration.
Additional samples from the citric acid series (Table 18) were ana-
lyzed after 336 hrs, or 336 hr with 72 hr additional exposure after add-
ing citric or acetic acid. These results are an indication of the com-
pleteness and reproducibility of the reaction. The results are shown
in Table 19.
-------
TABLE 19
ANALYSES OF INTEGRAL DDT-Zn- Cu-CITRIC ACID
SAMPLES AFTER REACTION
Analysis
Sample Exposure DDEt DDMS DDD DDT
336 hrs indoors 82.4 14.1 0.8 0.0
336 hrs out-of-doors 76.6 8. 1 7.4 0. 6
408 hrs indoors 84. 0 12. 9 0. 6 0.2
408 hrs indoors (336 hrs, then
add 1 g citric acid and 72 hrs
additional) 83. 9 13. 0 1.0 0. 0
408 hrs indoors (336 hrs, then
add 1 ml acetic acid and 72 hrs
additional) 84.9
Average 82.4
Calculated as equivalent % DDT from which component was produced.
A similar series of tests was carried out in which sulfamic acid was
employed as the acid source. These samples were prepared in the
same manner as the citric acid samples, excepting that the 4. 85 g
(50 millimoles) of sulfamic acid was added in 50 ml of water (instead
of the absolute ethanol used for citric acid), since sulfamic acid is
insoluble in ethanol and similar organic solvents. There was a rapid
bubbling and an odor of HgS following acid addition. The mix was
cooled in dry ice and stripped in a rotary evaporator; the operation
was difficult because of the gas evolution. The hygroscopic mass was
then ground and DDT added as before. The samples were than split
into several portions, moistened and exposed as before.
Analyses of the samples after 0, 24 and 168 hr reaction indicated that
91. 3, 74. 5 and 88. 0%, respectively, of the DDT remained in the three
samples. It was then found that both the acid and most of the metal
reductant had been consumed. It was first found that the extract from
the 1 week sample was neutral, rather than pH 2-4 as known to be neces-
sary for effective reaction. Hence, the evidence of little or no reaction
appears due at least in part to the consumption of the acid. Some of the
initial samples were treated with additional acid and tests were con-
tinued with no significant further reaction.
The experience with gas evolution from the reductant-acid mixture sug-
gests that it would be desirable to obtain information on the extent of
zinc consumption at various stages of the operation. Consequently, a
30
-------
device was set up to test evolution from the reductant residue by treat-
ing with hydrochloric acid and measuring the volume of hydrogen
evolved. The data from several tests follows:
TABLE 20
ZINC CONSUMED ON ACID COATING AND
AFTER REACTION WITH DDT
Unconsumed Zinc,
Sample %
Citric acid coated, before DDT application 82
Citric acid coated, after DDT application 57
Citric acid + DDT, 117 hr reaction 31
*Sulfamic acid + DDT, 168 hr reaction 19
*Sulfamic acid + DDT, 168 hr reaction 8
Sulfamic acid + DDT, 336 hr reaction 4
#
HS odor.
These results show that substantially all of the zinc had been consumed
in the sulfamic acid-coated samples tested, and that substantial reac-
tion appeared to have taken place during the coating operation with the
citric acid- treated samples.
IMPROVED CATALYST STUDIES
Studies were made of several systems that might give better catalysis
than the copper couple employed to improve the reduction with zinc,
aluminum, and iron. One hypothesis is that elements immediately to
the left of the reductant on the periodic table should form p-type semi-
conductors that would provide a catalytic surface (Reference 6).
On this basis, the efficiency of cobalt and nickel in catalyzing zinc,
aluminum, and iron was examined. Tests were carried out in which
1 meq of Cu**, Co**, or Ni** was added to 1 g of powdered reductant
to form the couple, 1 g of DDT was added in ZO ml of acetone, and the
solution was made acid with 10 ml of 1. 5 N acetic acid. Tests were
run for 24 hr at 25°C unless otherwise indicated.
The results with zinc reductant follow in Table 21.
31
-------
TABLE 21
EFFECT OF CATALYST ON ZINC REDUCTION OF DDT*
#*
Component Catalyst Couple:
DDEt
DBF
DBH
DDMU
DDMS
DDD
DDT
TTTB
DDA
B alance
Analysis , %, Following 24 hr
Reaction at 25°C
Ni
36.4
1.4
2.2
4. 7
13.7
37.2
0
3. 1
1.7
100.4
Co
28.6
1. 1
1.5
5.0
10. 6
35.7
0
8.9
1.2
92.6
Cu***
52.7
-
-
-
7. 1
30.0
0
0
0
94.9
Procedure employed was to dissolve 1 g DDT in acetone, then
add 1 g of zinc dust followed by 1 meq of the designated catalyst
ion to form the couple. The solution was made acid with 10 ml
of 1. 5 N acetic acid and stirred with a magnetic stirrer.
Calculated as equivalent % DDT.
###
A 2 hr reaction.
These results indicate that the copper couple formed in the same man-
ner as nickel or cobalt is clearly superior in that the reaction was more
rapid, and that the conversion to DDEt was more complete with less
DDD as an intermediate product. A noticeable warming of the reaction
flask was observed in the reaction with the Zn> Co couple.
Catalysis of aluminum by silver, nickel, cobalt, and zinc was also
attempted. Again the results were inferior to those obtained when the
copper catalyst was employed. The data are shown in Table 22.
32
-------
TABLE 22
EFFECT OF CATALYST ON ALUMINUM REDUCTION OF DDT*
Analysis , %, Following 24 hr
Catalyst
Component Couple:
DDEt
DBF
DBH
DDMU
DDE
DDD
DDT
TTTB
DDA
Minor Components
B alance
Reaction at 25 C
5.0
6.7
-
3.7
1.6
7.8
0.2
33.3
10.0
-
68.3
Co
0.6
0.3
-
-
3.5
1.0
93.0
0
0
-
98.4
Ni
0
-
-
3.1
0. 6
96.2
-
-
-
99.9
Zn
0
0.6
-
-
0.9
0. 7
82. 7
0.2
-
-
85. 1
Cu
0. 6
6.9
4.8
-
2.8
0.3
36.6
38.0
1. 1
91. 1
•j; !
Procedure same as Table 20 except aluminum powder reductant
instead of zinc dust.
3JC9JC
Calculated as equivalent % DDT.
72 hr reaction.
Attempted catalysis of iron reduction of DDT by cobalt or nickel
couples was likewise inferior to the results obtained with copper. The
results are given in Table 23.
33
-------
TABLE 23
EFFECT OF CATALYST ON IRON REDUCTION OF DDT*
*#
Analysis , %, Following 24 hr
Catalyst
C omponent C ouple :
DDEt
DBF
DDE
DDD
DDT
TTTB
DDA
Minor Components
B alance
Co
_
-
2.7
1.8
96. 1
2.9
-
-
103.5
Reaction at 25 C
Ni***
0.9
-
4. 1
3.8
73.2
15.2
-
-
97.2
Cu
0.6
1. 1
1.7
8.2
8.5
69.5
5.4
0.3
95.3
Procedure employed was to dissolve 1 g DDT in acetone, then
add 1 g of CP powdered iron followed by 1 meq of catalyst ion
solution to form the couple. The mixture was then made acid
with 10 ml of 1. 5 N acetic acid and stirred with a blade stirrer.
Calculated as equivalent % DDT.
*#*__ ,
25 hr reaction.
34
-------
SECTION V
CHARACTERIZATION OF DDT DEGRADATION PRODUCTS
Important to the development of a system for the controlled degradation
of field-applied DDT is an understanding of the physical and chemical
properties of the degradation products. This enables one to determine
whether the products are leached into runoff or ground waters, whether
the materials are vaporized from the field, whether the products can
be further reacted, are hydrolyzed by contact with water, etc. Accord-
ingly, a limited program of characterization of the principal products
from the degradation of DDT, DDEt and TTTB, was undertaken.
SOLUBILITY IN WATER
Solubility of DDEt in Water
The solubility of DDEt in water has been determined, essentially by the
method of Biggar, Dutt, and Riggs (Reference 8). Purified p, p1-DDEt
(25 mg) was dissolved in acetone and added to IX of water. The glass-
ware used in these tests had not been exposed previously to DDT or
degradation products and the water was deionized water that was addi-
tionally doubly distilled from glass. The acetone was removed by
evaporating about three times the acetone volume from the water by
heating on a steam-bath at 90-95°C for 20 minutes under a stream of
nitrogen while stirring with a magnetic stirrer. The suspension was
then cooled to ambient temperature, and the volume brought back to
li with water. It was then equilibrated with shaking in a 20 C constant
temperature bath for 1 to 6 days. The samples were filtered through
a fine (5/^.m) fritted glass filter, and the DDEt was extracted three
times with pesticide-quality hexane. The combined extracts were evap-
orated to a known volume (0. 5 - 1. 0 ml) and analyzed by gas chroma-
tography. The results of replicate tests are shown in Table 24.
TABLE 24
SOLUBILITY OF DDEt IN WATER AT 20°C
76
65
64
63
74
Mean 68
Standard
deviation 6.6
35
-------
This solubility is about 20-fold greater than the solubility of p,p'-DDT
reported by Biggar, et al. (Reference 8) (3. 4 ppb). The DDT solubil-
ity reported by Biggar is in substantial agreement with other determina-
tions summarized by Gunther (Reference 9).
The same basic technique was used in attempting to determine the solu-
bility of DDEt at 40°C. Purified p, p'-DDEt in acetone was added to
doubly-distilled water, and the acetone was removed by evaporation.
The samples were equilibrated with shaking in a 40 C water bath for
11 days.
Since the solubility of DDEt was expected to be somewhat greater at
40 than at ambient temperature, a method was required for removing
the excess DDEt from the solution at the equilibrated temperature so
that the temperature effect on solubility could be accurately determined.
Accordingly, a filtering device was prepared for removing the undis-
solved DDEt from the DDEt-saturated water while maintaining the tem-
perature of the solution at 40 . A filter stick (8 mm) with a fine (5/tm)
fritted glass filter element was fused to glass tubing to form a U-shaped
device that would filter directly from the thermostatted flask containing
saturated DDEt (+ excess product) into a filter flask. Nearly all of the
solution could be filtered in this manner while maintaining the solution
at temperature. The unfiltered residue (^5 ml) was then measured and
the solution volume corrected.
The DDEt was extracted three times with benzene, and the combined
extracts were dried over anhydrous Na2SO^, concentrated to a known
volume (1 ml), and analyzed by gas chromatography. However, com-
plex gas chromatographic records were obtained instead of the expected
single peak for DDEt. The presence of DBH was indicated in the sam-
ples (a few % of DDEt, exact amount difficult to establish) as well as
DDEt. It appears that the higher temperature exposure of DDEt to
water has resulted in hydrolysis of the compound.
Solubility of TTTB in Water
The solubility of TTTB was determined in a similar manner to the DDEt.
A portion of purified TTTB (ca 10 mg) was placed in a flask and doubly-
distilled water was added. Since TTTB was nearly insoluble in acetone, the
material was added directly to water and it was shaken until equilibrium
solubility was believed to be established. The samples were shaken in a
gyratory shaking bath at 40 C for 9 days. The samples were filtered
to remove undissolved TTTB by the method described for DDEt and the
TTTB was extracted three times with benzene. The combined extracts
were evaporated to dryness; no weighable residue was obtained (
-------
with a benzene -methanolic sodium methylate solution. Maximum absor
bence is at about 596 nm. The sensitivity of the method is about 1-2
or about 100-fold improved over the gravimetric method.
However, when the residues from the solubility tests were treated, no
significant difference from blanks were obtained, so that the solubility
of TTTB in water at 40°C appears to be < 1 ppb. Testing at 25°C was
therefore not attempted.
SOLUBILITY IN FATS
An important characterization of the products of DDT degradation is the
solubility in fatty tissues, since it is well known that DDT is concen-
trated through the food chain by virtue of its fat solubility. An indica-
tion of the solubility of the degradation products DDEt and TTTB in fats
could be established from measurements with the liquid fat triolein
(glyceryl trioleate). The solubility was established by incrementally
adding either DDT, DDEt, or TTTB to filtered triolein, stirring until
solubility was achieved, and adding further increments until the solu-
tion was saturated. The results of tests at ambient temperature (23-
25°C) follow in Table 25.
TABLE 25
SOLUBILITY OF DDT, DDEt AND TTTB IN TRIOLEIN
Material Solubility, %
DDT 13.0
DDEt 31.7
TTTB 0.27
TTTB* 0. 39
0. 5% lecithin added to triolein.
These results show clearly that the degradation product TTTB is but
slightly soluble in the fat, the solubility of the TTTB being 48-fold less
than DDT in triolein. This result is in line with the early claims that
TTTB would not have insecticidal properties because of limited fat
solubility (Reference 11). DDEt, however, has somewhat greater sol-
ubility than DDT in triolein. This result is not unexpected because of
the greater aliphatic nature of the DDEt molecule, compared to DDT.
SOLUBILITY OF TTTB IN VARIOUS SOLVENTS
While the product DDEt is freely soluble in a variety of organic solvents
(acetone, benzene, hexane, etc.), little is known of the solubility of
37
-------
TTTB in common, solvents proposed for use in its preparation and
purification. The results of a series of qualitative tests with a vari-
ety of solvents are presented in Table 26.
TABLE 26
SOLUBILITY OF TTTB IN VARIOUS SOLVENTS
*
Solubility of TTTB
Solvent Ambient (23-25°C) Hot
Methylene chloride si s
Chloroform s si s
Carbon tetrachloride si s
Benzene si s
Acetone i
Methyl ethyl ketone poor s
Ethyl acetate s
Ethanol i
Isopropanol i
Sec-butanol si s
Glacial acetic acid si s
Dioxan si s
Tetrahydrofuran (THF) s
Chlorobenzene s
#
s = soluble, si s = slightly soluble, i = insoluble
VAPOR PRESSURE
The vapor pressure is an important parameter in determining the per-
sistence of degradation products under field conditions, since a mod-
erately high vapor pressure may lead to volatilization of the products
from the soil, particularly in warm areas. A survey of available tech-
niques for determining the vapor pressure of pesticides revealed that
the gas saturation method employed by Spencer, et al, ^References 12,
13, 14} appeared best for the purposes of this study.* J The DDEt was
placed on a silica sand support by dissolving 1 g in acetone, adding the
acetone solution to washed sand (1 kg) and carefully evaporating the
solvent. The DDEt-sand was then placed in a gas saturation tube (4
cm dia x 48 cm long), and nitrogen carrier gas was then saturated with
the DDEt by slowly passing the gas through the saturator. The DDEt-
38
-------
laden gas was then passed through a gas washing bottle containing
pesticide-grade hexane, which scrubbed the DDEt from the nitrogen.
The hexane solution was then analyzed for DDEt by gas chromatography.
The gas was freed of hexane by passing it through a solid CC^-cooled
trap and the gas volume was measured with a standard wet-test meter.
Gas saturation was established from a constancy of measurement as
the flow rate was changed. The results of several tests are shown in
Table 27.
TABLE 27
VAPOR PRESSURE OF DDEt
Total
N, Volume
JL
Temp
°C
N2
Flow
Rate
ml/ sec
Vapor
Density
of DDEt
/tg/£N2
125
125
47
24
24
24
8.7
4.5
1.9
0. 197
0. 144
0.203
Apparent
Vapor Pressure
of DDEt
mm Hg
1. 5 x 10
1.2 x 10
1.5 x 10
-5
-5
-5
Linear
Velocity
Through
Saturator
cm/sec
0. 70
0.36
0. 15
In this treatment, the vapor pressure is calculated from the vapor
density by the ideal gas equation.
These, results show that the vapor pressure of DDEt is about 80-fold
greater than p, p'-DDT (1. 7 x lO'7 at 20°C, (Reference 15). The sig-
nificance of this can be seen if one calculates the evaporation rate of
DDEt as compared to DDT. The calculation of the evaporation rate of
a series of compounds from glass plates has been made by Hartley
(Reference 15) who found the rate of loss to be proportional to the
vapor pressure multiplied by the square root of the molecular weight
(diffusion process). A calculated comparison between DDEt and DDT
evaporation rate is given in Table 28.
TABLE 28
CALCULATED EVAPORATION RATES OF DDEt AND DDT
Substance
DDEt
DDT
Vapor Pressure
mm Hg
l,.4x 10
1. 7x 10
-5
-7
Predicted Rate of Loss
M from Acre of Glass Plate
251 7. 8x 10"2 Ib/day
355 1.2 x 10"3 Ib/day
39
-------
On the basis of this calculation, DDT applied at the rate of 1 Ib/acre
would last a calculated 830 days. However, 1 Ib of DDT degraded to
DDEt (0. 71 Ibs) would last only 9 days. It would therefore appear
that DDEt would be removed from the soil reasonably rapidly by
evaporation. It should be noted that tests in which DDT in soil, or
simulated field conditions, was degraded, the yield of DDEt, partic-
ularly at longer reaction times, has been lower than expected.
Vaporization of the DDEt product is believed responsible for the ap-
parent low yield observed.
STABILITY OF PRODUCTS TO HYDROLYSIS
The planned characterization of the products DDEt and TTTB includes
the investigation of the fate of these materials under acid or basic
hydrolysis conditions. These data are necessary in establishing what
will happen to DDT degradation products after they are formed in the
field.
In an initial experiment, 1. 75 JL of doubly-distilled water was saturated
with DDEt and filtered, and a 250 ml sample extracted to determine the
DDEt content. The remaining sample was split into two 750 ml portions,
one of which was acidified to pH 2. 2 with sulfuric acid, the other was
made basic with NaOH (pH 12. 0). These samples were placed in a 20 C
temperature bath (with shaking) and samples were withdrawn periodically.
The samples were then extracted, concentrated, and analyzed. The gas
chromatographs after 72 hrs showed no peaks other than DDEt at the
initial concentration for both acidic and basic treatments. However, it
should be noted that in the 40 C solubility measurement of DDEt, sev-
eral % DBH was shown, apparently as a result of hydrolysis.
Similarly, TTTB (10 mg) was placed in water either made alkaline or
acid (as above, pH 12. 0 and 2. 1, respectively), and equilibrated at
20 C. An extract of the samples revealed no products which would
respond to the gas chromatograph after 72 hrs at 20 C.
REDUCTION OF TTTB
Since TTTB appears as a significant product when either catalyzed alum-
inum or catalyzed iron reductants are used for degrading DDT, it was of
interest to determine whether this material could be further reduced by
either the Zn»Cu or Al« Cu system. In these tests, 1 g of TTTB in 20
ml acetone was reacted with 1 g of the reductant, and the solution was
acidified with 10 ml of 1. 5 N acid. Acetic acid was used to acidify the
Zn» Cu reaction and sulfuric acid was employed with the Al« Cu reduc-
tant. The reaction was carried out for 377 hrs (nearly 16 days) at
ambient temperature. The gas chromatographic analyses showed no
decomposition. Essentially complete recovery of unreacted TTTB was
also shown.
IDENTIFICATION OF PRODUCTS
While the principal products, DDEt and TTTB, were identified earlier
40
-------
(Reference 1), the identification and characterization of some of the
other major products have also been attempted.
Identification of PDA
In tests with the Al» Cu reductant system using acetic acid to provide
the requisite acidity, it was found that a substantial portion of the
product mixture was not represented either by the TTTB precipitate
or the products identified by gas chromatography analysis (DDEt,
DBMS, DDD, DDT, etc. ) of the ace tone-soluble wash of the mix.
Since this unknown represented a substantial product, its identification
was attempted.
Samples from the Al» Cu-acetic acid reduction mixture after 24 and
168 hr reaction by the usual method were examined (Table 29). The
residue from the ace tone-soluble fraction was first compared with the
analysis of components in this fraction by gas chromatography. The
residue was substantially greater than the sum of GC-responsive
components:
TABLE 29
RESIDUE AFTER EVAPORATION FROM
Al.Cu REDUCTION OF DDT
Sum of Gas
Residue After Chromatographic
Reaction Evaporation, Responsive Components,
Time, hrs % %
24 72.3 4. 9
168 44.2 10.7
The residue from the 24-hr reaction was then examined. It was found
that a hexane extraction of the acetone-soluble residue dissolved
27. 2% of the sample, leaving 41. 5% insoluble matter. The hexane
insoluble material was then dissolved in hot benzene and a product
was removed by crystallization. The product had a melting point of
167°C. The material formed did not give a gas chromatograph response
in 30 min.
A portion of the residue, crystallized from.hot benzene, was then mixed
with KBr and pressed into a cell for infrared analysis. The pellet was
examined in a Perkin-Elmer Model 137B spectrophotometer. Absor-
bance was shown at 5.88, 6.74, 9.20, 9.89, 12.20, 12.40, 13.30,
13. 55 and 13. 80 /dm. Tentative constituent group assignments were
then made (Table 30).
41
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TABLE 30
INFRARED SPECTRA OF PRODUCT FROM
Al. Cu REDUCTION OF DDT
Wavelength,
Assignment
5. 88 Carbonyl
6. 74 p-Cl phenyl
13-14 Chlorine
A. peculiar "smeared-out" absorbance was also shown in the 3-4ytcm
range. Upon examining the spectra tabulated by Burchfield and Johnson
(Reference 16), the same absorbance was shown by DDA. When the
spectra above were compared with authentic DDA, idential infrared
spectra were obtained. A mixed melting point of the product obtained
from the reaction with authentic DDA showed no depression of the melt-
ing point, demonstrating that the product obtained was DDA.
Preparation of DDNU
A suspected unidentified product of DDT degradation was the ethylenic
analogue of DDEt, DDNU or 1,1-bis (p-chlorophenyl) ethylene. This
product was generally expected under the conditions which yield DDE.
The material was prepared and its gas chromatographic response noted.
The material was prepared basically by the method of Grummitt (Ref-
erence 17). A sample of commercial grade di(p-chlorophenyl)methyl
carbinol (Sherwin-Williams Dimite) was dehydrated to produce the
desired compound. The material was heated at 210 C for 15 min and
then was refluxed for 1 hr with 20% sulfuric acid. The crude product
was washed with ice water, dissolved in ethanol, and decolorized with
charcoal. The crystals had a melting point of 81. 5-84. 5°C. Upon
recrystallization from isopropanol and decolorization with charcoal,
colorless crystals with an 84-85 C melting point were obtained. The
value compares well with the 84-86°C melting point reported by
Grummitt (Reference 17), and 84-85. 5°C reported by Garbisch (Ref-
erence 18).
Identification of 4, 4'-Dichlorobenzophenone {DBP}
A further product suspected on the basis of unidentified gas chromatog-
raphy peaks was 4, 4'-dichlorobenzophenone. The identification con-
firmed its presence as a product (^^1%) in the Al« Cu and Fe« Cu reduc-
tion of DDT.
This identification was confirmed by isolation of the material and mea-
suring the melting point of the product and derivative. The dichloro-
benzophenone was obtained (by crystallization from ethanol) from the
42
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residue remaining after removing the solvent from the acetone-soluble
reaction products of an Al. Cu reduction of DDT. This material had a
melting point of 143-145 C, compared to literature values ranging
from 142 to 148 C, but mainly about 145°C (Reference 19). The 2,4-
dinitrophenyl-hydrazone derivative was then prepared by the method
of Haller, et al. (Reference 20), yielding orange crystals with a melt-
ing point of 237-239 C (Haller reported 238-240 C). It is therefore
concluded that the product is 4, 4'-dichlorobenzophenone.
PREPARATION OF MATERIALS
The quantities of DDEt and TTTB required for the fish toxicity testing
(Section VI), as well as physical property characterization and chemi-
cal stability measurements required that these materials be prepared
in pure form in moderate quantity.
Preparation of DDEt
In the initial attempt, the method of Becke and Buckschewski (Reference
21) was employed. In the attempt, chlorobenzene and par aldehyde were
condensed in the presence of anhydrous A1C13 and dimethylformamide,
with introduction of dry HC1. The product was fractionally distilled in
vacuum. However, gas chromatography of a solution of the product
showed that the distillate was a mixture of isomers and other impurities.
Further attempts at distillation and crystallization did not yield a puri-
fied form of the p, p'-DDEt.
Better results appear to have been obtained from a forced reduction of
DDT. In this preparation, 100 g of DDT was treated with 150 g of
copper-catalyzed zinc over a period of 68 hrs; the reactant mixture was
0. 5 N in sulfuric acid. Following the addition of the sulfuric acid, the
temperature rose to 49 C, but gradually fell to ambient (A/ 25 C). The
excess reductant was filtered off and washed with acetone. The acetone
was removed by evaporation and the oily residue was extracted with
benzene. Stripping of the benzene left 71 g of crude DDEt. Gas chroma-
tography of this fraction showed it to be mainly DDEt with about 3%
DDMS and DDMU, and 0. 1% DDD and DDT. This material WJLS then
vacuum distilled with the major product distilling at 150-152 C at 1-1.2
mm pressure. The DDEt upon recrystallization from methanol yielded
a product which was free from impurities as shown by the gas chroma -
tograph. The melting point, 54-55°C (corrected) is the same as that
reported by Grummitt, et al. (Reference 17).
Another batch of DDEt prepared by basically the same method showed the
presence of small amounts of DDM, DDE, and DDMS by gas chromatog-
raphy. Successive recrystallizations tended to diminish the amount of
DDMS substantially, whereas contamination with DDE was reduced rela-
tively little.
Further purification was required for the fish toxicity study, and removal
of DDE was considered necessary. Column chromatography was there-
fore attempted in order to remove the DDE. Columns employing silica
43
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gel, alumina, Florisil (an absorbent consisting primarily of SiO?
(84. 0 ± 0. 5%) and MgO (15. 5 £ 0. 5%); The Floridin Co. ), and Sephadex
(a partially alkylated, cross-linked dextran; Pharmacia Fine Chemicals,
Inc. ) were evaluated. The latter material, Sephadex, is a gel-filtration
medium modified for use with polar organic solvents, and capable of
separating components based on differences in molecular size.
One percent solutions of DDEt were employed as column feed. Hexane
was used as the solvent with silica gel, alumina, and Florisil, and
ethyl acetate with Sephadex. The ratio of absorbent to DDEt was 15:1
for all adsorbents except alumina, where a 7:1 ratio was used due to
column dimensions. Treatment with the Sephadex resulted in complete
removal of DDE, but had no effect on DDMS or DDM; furthermore, the
eluate contained an additional impurity (of very short retention time),
the nature of which is not known. Silica gel and Florisil both removed
DDE, but neither removed DDD or DDMS. Alumina adsorbed DDE and
DDMS, but had little effect on DDM. When the DDEt eluate from the
alumina column was freed of solvent and the DDEt recrystallized once
from methanol; the resulting DDEt was found to be 99. 9% pure, the only
contaminant being 0. 13% of closely related DDM.
In an attempt to simplify the preparation process, a batch of DDEt was
prepared by essentially the method used in previous preparations. That
is, a 100 g lot of recrystallized DDT was reduced with 500 g of copper-
catalyzed zinc to form the crude DDEt. The sample was reacted for
about 48 hours (10 hours at reflux, the remainder at 23-25 C), and
frequent samples were withdrawn for analysis. Analysis of the crude
acetone solution at the end of reaction indicated 89. 5% of the DDT had
been converted to DDEt, 3. 6% to DDMU, 3. 0% to DDMS, and the
remainder to minor products. Removal of the acetone, extraction with
benzene, and re crystallization from cold methanol led finally to a
crystalline •white product with a melting point of 53-55 C. The gas
chromatographic spectra indicated a DDEt purity of > 99% with the only
impurity being DDMS. This procedure was used in preparing DDEt used
in the fish toxicity testing.
Samples had been withdrawn from the batch during preparation in order
to determine whether additional reduction with fresh zinc-copper couple,
or Raney nickel, would remove the residual DDMU and DDMS found in
the crude preparation. However, neither treatment was successful.
Further examination of test data suggested that the reduction of DDD
rather than DDT might lead to a simpler preparation of pure DDEt
with less DDMS impurity. In a test, 10 g of DDD in acetone was reacted
with 50 g of zinc catalyzed by the slow addition of 50 meq of copper ion
(i. e., normal ratio of 1 meq copper ion/g zinc). The reaction was made
0. 5 N in sulfuric acid and was reacted at ambient temperature for 17 hr.
After removal of the acetone, extraction with benzene, and recrystalli-
zation from cold methanol, a yield of 6. 9 g of DDEt (theoretical yield
7. 8 g) was obtained. The gas chromatographic analysis showed that the
material was of better quality than the DDT-based preparation, with
only a trace impurity («0. 1%) of DDMS in the DDEt. Following this
44
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preparation, a 10-fold larger batch was prepared (100 g of DDD reacted).
This batch gave an 80% yield of DDEt, with the only impurity shown by
the gas chromatograph being <0.2%DDMS.
In another laboratory test, the reduction of DBA by Zn» Cu couple was
attempted as a possible means for producing DDEt without DDMU or
DDMS contamination. While some DDEt was produced (/^12% DDEt,
5% DDM), the low yield suggests the procedure is impractical.
Preparation of TTTB
In view of the early experience of Bernimolin (Reference 22) and
Riemschneider (Reference 11), where insecticidal effects at first attri-
buted to TTTB were found to be associated with DDT impurities in the
product, it was hoped that a preparation that did not involve DDT could
be used. However, an examination of methods of preparation did not
disclose a practical method of preparing this material that did not
involve DDT. The method selected was the Al« Cu-H2SO4 reduction of
DDT, known from previous data to give a high conversion to TTTB,
followed by recrystallization until the sample would show no trace of
DDT upon gas chromatographic analysis.
A 5 g sample of DDT was reacted in 100 ml of acetone with 5 g Al« Cu
couple to which 50 ml of 1. 5 N I^SC^ was added to provide the requisite
acidity. The reaction was then carried out for 48 hrs at ambient temper-
ature. The temperature rose from 21 C to 30. 5 G (about 3 min after the
inception of reaction), but returned to nearly ambient conditions within
40 minutes. After the 48-hr reaction period, the crude TTTB and
unreacted Al» Cu were separated from the remainder of the reactants by
centrifugation. The crude TTTB (+ Al« Cu) was extracted four times
with acetone, which removed most of the DDT. The TTTB was then
extracted with hot CHC13 and the solvent removed, leaving 3. 80 g (76%
yield) of TTTB. The partially purified TTTB was recrystallized from
tetrahydrofuran, and placed in a micro Soxhlet extractor and extracted
with boiling acetone. Three extractions with acetone were made, and
a decrease in impurities was noted in the gas chromatographic trace
after each extraction. Final purification was achieved by re crystalliza-
tion from ethyl acetate. A portion of the final product was dissolved in
benzene and analyzed by the gas chromatograph; no residual DDT, DDD,
nor other degradation products of DDT were found. Based on the quan-
tities employed, the level of DDT, DDD, or similar impurities must be
less than 0. 001% in the product. The melting point of the product was
268-270°C. A scale-up of this basic method was used for the preparation
of larger batches of TTTB employed in fish toxicity testing. :
45
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SECTION VI
TOXICITY TESTING OF DDT-DEGRADATION PRODUCTS
The second phase of the program involved studies designed to assess
the toxicity of DDEt and TTTB to selected aquatic organisms. One of
the objectives was to determine, by means of acute bioassay tests,
the 96-hr median tolerance limit (TLm) of both the above products to
three fish species and to one aquatic invertebrate which would be
representative of a fish food organism. The second objective was to
determine, by means of long-term continuous exposure studies, the
maximum acceptable DDEt and TTTB concentrations for the fathead
minnow. The maximum acceptable concentration would be estimated
on the basis of data collected on growth, survival, and reproductive
capacity of the test organism. A third objective was to utilize the
data collected from both types of tests on the fathead minnow for the
calculation of application factors for both products and to utilize these
application factors for predicting the maximum acceptable DDEt and
TTTB concentrations for the other two species of fish involved in the
study. The fathead minnow was selected as the basic test organism
because of its wide use as a reference test fish in toxicity testing.
The method used for predicting safe toxicant concentrations was pro-
posed by Mount and Stephans (Reference 24). The method requires an
estimate of the 96-hour TLm and the maximum acceptable toxicant
concentration (MATC) of a given toxicant for a reference fish species
(in this case the fathead minnow). The application factor is calculated
by dividing the MATC by the value for the 96-hr TLm. The MATC of
the same toxicant for a second fish species can then be estimated by
multiplying the 96-hr TLm concentration of the toxicant for the second
fish species by the application factor.
EXPERIMENTAL CONDITIONS
Test Animals
The three species of fish selected for the study were the fathead minnow
(Pimephales promelas, Raphinesque), the bluegill (Lepomis machrochirus,
Raphinesque) and the rainbow trout (Salmo gairdnerii, Richardson). Fat-
head minnows of mixed ages were purchased from Jim's Sportshop in
Gilroy, California, and shipped by air carrier to the El Monte laboratory.
Two purchases of approximately 800 fish each were made, the first on
January 20, 1971, and the second on May 18, 1971. The fish purchased
were sexually immature and measured approximately 2 in. in length.
The minnows were acclimated in the laboratory for at least two weeks
before being used in any test. During the acclimation period, they were
kept in round polyethylene tanks containing about 20 gallons of water
which was changed continuously and held at 23 +. 1 C. During the first
week after acquisition, the minnows were treated with tetracycline-HCl
47
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(Richlyn Labs, Inc. , Philadelphia) to minimize bacterial infections.
Tetracycline was administered at a rate of 250 mg per five gallons of
water. The water was kept static during treatment. All fish were fed
Oregon Moist Trout Pellets (R. V. Moore, Inc. , LaConner, Washington),
ad libitum, once a day except on weekends.
The bluegill and rainbow trout were purchased from Corcoran Brothers
of Azusa, California, and brought to the laboratory by truck. The blue-
gills were purchased on 27 October 1971, and the trout on 10 November
1971. Both fish species were acclimated in the laboratory for at least
two weeks before being used in any test. During the acclimation period,
the fish were kept in 15-gal aquaria containing about 10 gal of water.
The water was changed continuously and maintained to within^ 1 degree
at 12 C and 23 C for the trout and the bluegill, respectively. These
fish were not treated with antibiotic. The bluegills were fed ad libitum
once daily on Oregon Moist Trout Pellets and the trout were fed Purina
Trout Chow in a similar manner.
Daphnia magna were obtained from the National Water Quality Labora-
tory in Duluth, Minnesota, in January 1971, and propagated in the
Envirogenics Systems Company Aquatic Biology Laboratory. These
microcrustaceans were kept in 15-gal aquaria and fed compressed yeast
supplemented by phyto- and zooplankton cultured in an infusion made
from filtered pond water and horse manure. >
Laboratory Water Supply
The source of the water used in the laboratory was the El Monte Munici-
pal water supply, obtained from local wells. Water entering the labora-
tory was first passed through activated charcoal columns (Sparkletts
Water Co. ) and then through an electronic liquid sterilizer (Aquafine
Corp., Model SL-1, Los Angeles). The sterilizer is an ultraviolet
light equipped with a 260 nm wavelength monitor and alarm system. The
purpose of the sterilizer was two-fold. One was to convert any residual
chlorine present as hypochlorite to gaseous Cl2 for later air stripping.
The second objective was to reduce the number of microorganisms that
might have entered with the water.
A portion of the treated water was piped through a stainless steel heat
exchanger operating off of a 250-ton plant refrigeration system. This ,
system was designed to provide chilled water to the test laboratory.
Water leaving the heat exchanger, as well as the bypass water from the
sterilizer, was piped into head tanks located within each test laboratory.
The heat tanks, constructed of acrylic plastic (Plexiglas), were equipped
with stainless steel temperature-controlled heating units. The heating
units were used only when necessary. Each head tank was also equipped
with a spinning-disc aerator. The disc aerator consisted of about 75
mylar discs measuring 4 in. in diameter, mounted 1/4 in. apart on a
stainless steel rod by means of polyethylene spacers. The device was
mounted horizontally across the head tank so that one-half, of each disc
was exposed to air. The shaft was then rotated by an electric motor .
48
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equipped with a speed control. The device provided water saturated
with oxygen to the diluters.
Each head tank was designed to deliver water to a Mount-Brungs pro-
portional chemical diluter (Reference 23) and to a manifold constructed
of polyvinyl chloride plastic tubing. The manifold permitted delivery
of water to the individual test tanks when necessary.
The results of a chemical analysis of the water are shown in Table 31.
ACUTE TOXICITY STUDIES
Acute Toxicity Studies on Selected Fish Species
Experimental Design
These studies were designed to provide data for estimating the 96-hr
median tolerance limit (TLm) of DDEt and TTTB for the fathead min-
now, bluegill, and rainbow trout. The bioassay procedures used in
these studies were those of Standard Methods (Reference 25), modified
slightly.
Acute bioassay tests, designed to determine the 96-hr TLm concentra-
tion of DDEt for fathead minnows, were conducted in static test solutions,
and the effects of aeration and DDEt-dispersing agents were evaluated.
Initial concentrations of DDEt as high as 100 ppm were used. Acute
tests involving TTTB and the fathead minnow were carried out only with
TTTB-saturated water; dispersing agents to increase the amount of sus-
pended TTTB were not used. All test fish were fasted for 24 hr prior
to testing and food was withheld throughout the duration of each test.
The acute tests with bluegill and rainbow trout involved the use of a
flow-through toxicant delivery system designed to achieve continuous
renewal of the test medium. The tests were conducted in 1 x 1 x 2 ft
(HWLi) glass aquaria outfitted with drains which allowed the tanks to
fill to a depth of 10 in. At this depth each tank held about 50 liters of
test solution.
Laboratory water was delivered to each test container at a rate of 200
ml per min, providing a turnover of approximately 5. 75 tank volumes
per day. Water temperatures were maintained at 12 +_ 1 C and 23 +^ 1 C
for the trout and the bluegills, respectively.
Acute toxicity testing with the bluegill and trout were conducted at one
concentration only -- the saturation level of DDEt or TTTB in water,
since earlier tests with'the fathead minnows indicated that the toxic
limit would probably be no lower than saturation. These values were,
respectively,-^ 0. 05 and ^0. 001 ppm.
Each toxicant was dissolved in acetone before being mixed with the
stream of water being delivered to the test container. Delivery of the
acetone solution was accomplished by using a Sage syringe pump
49
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TABLE 31
Total Hardness
(as CaCOj
Calcium
Magnesium
Potassium
Sulfates
Sulfides
Nitrates
Nitrites
Ammonia
Total Alkalinity
pH
Specific Conductance
Chlor amines
ALYSIS OF RAW WATER SOURCE
SH BIOLOGY LABORATORY
m^
148
40.
11.
0.
7.
<0.
<0.
0.
0.
!
-------
(Model 255-1) fitted with a 50 cc syringe. The concentration of DDEt
in the test tank was determined by analyzing samples of the test solu-
tion by gas chromatography, after hexane extraction and concentration
in a Kuderna-Danish evaporator. The concentration of the saturated
TTTB solution was estimated on the basis of water delivery rate, the
concentration of the TTTB-acetone solution, and information previously
obtained on the solubility of TTTB in water; the concentration of TTTB
in water was too low to measure accurately by any available means.
The survival of each fish species in DDEt- or TTTB-saturated water
was compared with the survival of the same species in unadulterated
laboratory water. Each test was conducted once, and the duration of
each test was 96 hrs. Food was withheld for 24 hr prior to testing and
during the tests. The number of trout and bluegill tested per tank were
5 and 10, respectively. The mean weights and lengths of the test fish
are listed in Table 32.
TABLE 32
MEAN WEIGHT AND LENGTH OF TEST BLUEGILL AND
TROUT USED IN ACUTE TOXICITY TESTING
Controls Toxicant Exposed
Toxicant/Fish Species Weight Length Weight Length
(g)(mm) (g)(mm)
DDEt/bluegill 1. 73 49. 6 1. 66 48.5
TTTB /bluegill 1.94 51.8 1.75 50.6
DDEt/trout 31.5 154 31.3 156
TTTB/trout 34.2 152 34.5 152
Data and Discussion
Test mixtures, in which up to 100 ppm DDEt were added, were prepared
by mixing measured amounts of DDEt in acetone with two liters of water.
These mixtures had no toxic effect on fathead minnows exposed for 96
hours. Analysis of filtered samples of the test mixtures showed DDEt
to be present in solution at concentrations no higher than 0. 05 ppm. The
remaining DDEt added was precipitated. It was observed that mixtures,
ma'de to contain DDEt in'concentrations exceeding 0. 05 ppm, contained
relatively large particles of precipitated DDEt on the surface of the
water. A fresh set of the same mixtures were prepared and gently
aerated to keep the undissolved particles more evenly distributed. As
in the previous test, 2 minnows were placed in each test container
(only 2 fish were used since this was a screening test). The test results
were the same as with the unaerated mixtures; no toxic effects were
noted on the fish.
51
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These tests indicated that the 96-hr TL.m concentration was greater than
0. 05 ppm (or saturation) and that a method of preparing stable suspen-
sions of DDEt was needed.
Exploratory tests with two eznulsifiers were conducted. An acetone solu-
tion of DDEt was mixed with Triton X-100, a dispersing agent recom-
mended by Mount and Brungs (Reference 23), to form a suspension con-
taining 1. 56 ppm DDEt and 1. 88 ppm Triton X-100. The suspension was
allowed to stand undisturbed for 48 hr. During this time, most of the
DDEt had settled and samples taken above the residue contained no more
than 0. 05 ppm DDEt. Fish exposed to this suspension for 96 hr survived
well.
Further tests were conducted using an agent commonly employed in pre-
paring emulsifiable concentrates of DDT. This agent was Agrimul
(Napco Chemical Company), a non-ionic ether-linked dispersant. This
dispersant was evaluated both with and without added aromatic solvent.
In the first test, 6. 3 mg of Agrimul and 1. 0 ml of acetone containing
15 mg of DDEt were mixed; then 100 ml of water was added and the mix-
ture shaken to disperse the DDEt. The emulsion was then added to 2525
ml of water, mixed and allowed to stand. A second mixture was pre-
pared in a similar manner except that 1. 0 ml of xylene was added
(xylene is commonly used in field-applied formulations). After both
mixtures had stood for 24 hr, they were still quite cloudy, indicating
that both contained a substantial amount of DDEt in suspension. A 1.0.?.
sample was withdrawn from each mixture, extracted 3 times with hex-
ane, concentrated to 1. 0 ml and analyzed by gas chromatography.
In the preparation without xylene, 2. 6 mg/JL, or 46% of the initially added
DDEt remained in suspension. In the preparation containing xylene, 5. 1
mg/JJ,, or 89% of the initially added DDEt remained in suspension. These
experiments demonstrated that Agrimul 70A aided substantially in keep-
ing DDEt in suspension and that xylene strikingly augmented this effect.
Bioassays on these mixtures followed. The xylene mixture, containing
2. 2 ppm Agrimul and 310 ppm xylene killed all of the test fish within
30 sec. On the other hand, the mixture without xylene, and with the
same Agrimul content, did not affect any. of the test fish during a 6-day
exposure period. The toxic effect observed in the first test was deemed
due to the xylene.
Accordingly, fish were then exposed to an aqueous suspension containing
2. 2 ppm Agrimul and 10 ppm DDEt for 6 days. At the end of the exposure
period, all of the fish were alive and showed no untoward behavior.
Aaalysis of the suspension after 23 hours showed that 6 ppm DDEt (60%)
remained in suspension at the conclusion of the test. Another bioassay
was conducted on a suspension initially containing 2. 2 ppm Agrimul and
100 ppm DDEt. The mixture was very milky in appearance. A single
fish placed in this suspension was alive after 6 days of exposure. However,
after 72 hr, the fish lost and did not regain its equilibrium. Analysis of
the suspension after 71 hr showed that 16. 1 ppm DDEt remained in sus-
pension. It appeared that most of the DDEt was precipitated early in the
52
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test so that the time-average concentration was probably near 16. 1 ppm.
Further attempts to determine the 96-hr TLm of DDEt for the fathead
minnow were abandoned. Tests conducted demonstrated that exposure
of the minnows to a saturated solution of DDEt (50 ppb) for 96 hr was
not harmful. Addition of Triton X-100 or Agrimul 70-A helped to keep
DDEt in suspension, but the amount maintained in suspension was
neither consistent nor long-lasting. Although the fish exposed to a sus-
pension of Agrimul and 16 to 100 ppm DDEt were affected, the question
remained as to whether loss of equilibrium was due to DDEt alone or
the extreme turbidity of the suspension.
Attempts to determine the 96-hr TLm of TTTB for fathead minnows were
not made; tests were run only at saturation where no effect was noted.
Subsequent data from acute tests on TTTB with bluegills and rainbow
trout, as well as results obtained from the chronic studies on TTTB with
fathead minnows, suggest that if minnows were exposed to a saturated
solution of TTTB { ^ 1 ppb), for 96 hr, they would not be adversely
affected.
The data in Table 33 indicate that, like the fathead minnow, the blue gill
and rainbow trout can survive well in DDEt- and TTTB-saturated water
for 96 hr. No toxic symptoms were observed.
TABLE 33
MORTALITY AFTER 96 HOURS EXPOSURE
Mortality/No. Exposed
Toxicant (Cone. ) Fish Species Control Toxicant Exposed
DDEt (50 ppb) Bluegill 0/10 0/10
TTTB «1 ppb) Bluegill 0/10 0/10
DDEt (50 ppb) Rainbow trout 0/5 0/5
TTTB (<-! ppb) Rainbow trout 0/5 0/5
The reason for limiting the maximum test concentrations of DDEt and
TTTB to 0. 05 and ^0. 001 ppm, respectively, in the tests utilizing
the bluegill and the rainbow trout was two-fold. First, earlier attempts
at preparing uniform suspensions of DDEt containing greater than sat-
urated concentrations met with failure. TTTB is only slightly soluble
in acetone, which is considered a reasonably non-toxic organic solvent
having a 96-hr TLm of about 10, 700 ppm (Reference 24). To attempt
to develop a method for satisfactorily maintaining both compounds in
suspension would have been time-consuming. Secondly, toxicity data
from tests involving the use of solvents and dispersants_in combination
with the test compound are often meaningless. Often, the observed
toxic effects are due almost entirely to the solvent or emulsifier
53
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(Reference 26). Workers at the Duluth EPA laboratory recommended
against attempting to run acute toxicity tests at concentrations greater
than the solubility of the toxicant in water.
DDEt is no more than and apparently much less than half as acutely
toxic to fathead minnows, bluegills, and rainbow trout as DDT.
According to Henderson and co-workers (Reference 27), the 96-hr
TLm concentration of the 100% p, p'isomer of DDT for fathead minnows
in soft and hard water is 0. 026 ppm. Tests on the technical grade of
DDT gave 96-hr TLm estimates of 0. 021 ppm for bluegills, 0. 042 ppm
for fatheads, 0. 036 ppm for goldfish, and 0. 056 ppm for guppies. The
96-hr TLm concentration of an unspecified grade of DDT for rainbow
trout is reported in Water Quality Criteria (Reference 28) as ranging
from 0. 024 to 0.074 ppm. Since the solubility of TTTB in water is
only about 0. 001 ppm or less, no comparison can be made with this
toxicant.
Since TLm estimates on DDEt and TTTB for the three species of test
fish could not be obtained, the third objective, that of calculating the
maximum acceptable DDEt and TTTB concentrations for rainbow trout
and bluegills, could not be stated. Indeed, the TLm of DDEt for the
fathead minnow, blue gill, and rainbow trout can only be stated as
> 0. 05 ppm, and for TTTB,> . 001 ppm.
Acute Toxicity Studies on Daphnia Magna
Experimental Design
These tests were designed to provide data for estimating the 96-hr TLm
of DDEt and TTTB for the microcrustacean, Daphnia magna. For com-
parative purposes, similar tests were conducted on DDT.
The maximum concentration of each compound tested was that obtainable
at saturation at room temperature, which ranged from 22-24 C. Satu-
rated solutions were prepared by shaking an excess amount of the com-
pounds in 2 liters of laboratory water for 48 hr and filtering off the
undissolved material. Gas chromatographic analysis of samples showed
46-55 ppb DDEt and 0. 9 ppb DDT, respectively. The TTTB concentra-
tion from solubility tests was estimated to be 1 ppb or less.
The first series of tests was carried out on DDEt and DDT. Ten daphnids,
less than 24 hr old, were placed in 400 ml beakers containing 200 ml of
unaerated water with different amounts of DDEt or DDT. Every 24 hr,
for 96 hr, the dead daphnids were removed and counted. Since it was
occasionally difficult to determine if a carcass was actually a dead daphnid
or a cast, the survivors were also counted. Food was withheld during
the tests. The tests were repeated once. The data are shown in Tables
34 and 35.
54
-------
TABLE 34
CUMULATIVE PERCENT MORTALITY OF
D. MAGNA EXPOSED TO DIFFERENT
CONCENTRATIONS OF DDEt AND DDT (Test 1)
Mortality % after Test, hr
Cone, (pj
0
0
0.09
0.9
9.2
23. 0
46.0
>b) 24
0
0
10
10
10
0
0
DDEt
48
0
0
20
20
10
20
30
72
50
0
30
50
10
40
50
DDT
96 Cone, (ppb)
50
40
50
60
20
50
60
TABLE 35
0
0
0.0009
0.04
0.09
0.45
0.90
24
0
0
0
0
0
0
0
48
10
0
30
0
10
40
80
72
40
20
30
30
20
100
100
96
60
20
40
40
40
100
100
CUMULATIVE PERCENT MORTALITY OF
D. MAGNA EXPOSED TO DIFFERENT
CONCENTRATIONS OF DDEt AND DDT (Test 2)
Mortality %
after Test
, hr
DDEt
Cone, (pj
0
0. 09
0.9
9.2
23.0
46.0
>b) 24
0
0
0
0
0
0
48
0
0
10
10
20
0
72
0
0
10
10
30
0
96 Cone, (ppb)
30
0
20
10
30
0
0
0. 009
0.04
0.09
0.45
0.90
24
0
0
0
0
0
10
DDT
48
10
10
10
30
0
60
72
50
10
30
30
10
70
96
100
70
50
50
10
80
A second series of tests, utilizing the same test concentrations, was
carried out subsequently. In these tests, the test solutions were
renewed every 24 hr and 1 ml of an 0,2% yeast suspension added as
food. The data are shown in Table 36.
55
-------
TABLE 36
CUMULATIVE PERCENT MORTALITY OF D. MAGNA
EXPOSED TO DIFFERENT CONCENTRATIONS
OF DDEt AND DDT AND FED YEAST
After Test, hr
DDEt
24
0
0
0
0
0
10
48
0
0
0
10
20
10
72
20
0
0
10
30
10
96
20
0
0
20
30
10
Cone.
0
0.
0.
0.
0.
0.
(ppb) 24
009
045
09
45
90
10
0
10
0
20
0
DDT
48
10
0
20
0
20
0
72
10
30
30
0
20
0
10
30
40
0
30
10
Cone, (ppb) "24 48" "72" "W
0
0.092
0.92
9.2
23.0
46.0
1. 0 ml 0. 2% yeast suspension per day, flow-through system.
Tests were also conducted on various dilutions of DDEt- and TTTB-
saturated water with mature daphnids as test animals. Test conditions
were the same as those used in the first test series; however, the
length of the tests was shortened to 48 hr; no census was taken until
the 48th hr and 20 animals were exposed to each concentration.
Data and Discussion
Mortality of the yound daphnids exposed to DDT or DDEt was inconsis-
tent (Tables 34, 35, and 36). Mortality among controls was Unusually
high, and in some cases even greater than that of the daphnids exposed
to the highest toxicant concentrations. The addition of food and renewal
of the test solutions lowered mortalities in most cases, but did not
improve the consistency of the mortality rate.
Tests on mature daphnids provided considerably more consistent data
(Table 37). For DDEt the dose-response curve (Figure 1) was essen-
tially linear between 13. 2 and 55 ppb. The 48-hr TLm was estimated
at 35 ppb. The toxicity of DDEt and DDT to Daphnia appears similar.
Anderson (Reference 29) reported that D. magna, exposed to DDT con-
centrations ranging from 1 to 100 ppb, were immobilized in periods
ranging from 16 to 32 hrs. Frear and Boyd (Reference 30) estimated
the 26-hr TLm concentration of DDT for D. magna as 4. 4 ppb. For
D. pulex, the 48-hr TLm concentration of DDT was estimated at 36 ppb
"(Water Quality Criteria, Reference 31).
56
-------
TABLE 37
PERCENT MORTALITY OF ADULT D. MAGNA
EXPOSED TO DDEt OR TTTB FOR 48 HOURS
DDEt
Cone, (ppb)
0. 00
2.75
7.43
13.2
23.05
41.2
55. 0
% Mortality
5
0
0
10
25
60
90
TTTB
Cone, (ppb)
0. 00
0. 05
0. 14
0.24
0.42
0. 75
1.0
% Mortality
10
0
0
0
5
5
0
CHRONIC TOXICITY STUDIES
Chronic Study with DDEt-Saturated Water
This study was originally started with four DDEt concentrations in
addition to control. Except for control, the least concentrated test
solution contained approximately 0. 05 ppm DDEt which is approximately
the solubility limit of DDEt in water at 20-25 C. After the tests were
underway, problems in maintaining DDEt in suspension at levels exceed-
ing saturation made it necessary to abandon tests on concentrations
exceeding 0. 05 ppm.
The study on the long-term effects of DDEt-saturated water on growth,
survival and reproduction of the fathead minnow was continued. During
the investigation which was initiated oh March 24, 1971, the parental
fish were exposed continuously to DDEt-saturated water for a period of
28 weeks. A description of the equipment and procedures employed fol-
low.
The experiments and equipment were designed according to recommenda-
tions provided by the EPA Laboratory in Duluth, Minnesota. The testing
facilities permitted continuous renewal of the test solutions in the test
containers. Both control and DDEt-exposure tests were conducted in
duplicate.
The test tanks were placed on a double-shelved metal rack with the
larval tanks above the adult tanks, and were illuminated by two-tube
sets of 4-ft Durotest "Optima" fluorescent lamps set 18 in. above the
tanks. A plastic mixing cell, 4x2x6 in. high, was positioned above
each pair of larval tanks to receive the treated tapwater used in the
tests. Each mixing cell contained two outlets designed to distribute the
57
-------
100
80
c
S 60
&
1
40
-FI*a-
fit:
20
10
20 . 30 40
DDEt Concentration, ppb
50
60
Figure 1. Dose-Response Curve for Daphnia magna exposed to
Solutions of DDEt for 48 hours
58
-------
water evenly between each of the duplicate larval tanks. Water flowed
from the larval tanks into the lower adult tanks and then into a drain.
The depth of the water in all of the tanks was 6 in. At this depth, the
larval tanks held about 14 Ji of water and the adult tanks twice as much.
The water delivery rate to each larval-adult tank set was 200 ml per
min. The turnover rate for the larval and the adult tanks was about
10 and 5 tank volumes per day, respectively.
DDEt-saturated water was prepared by metering an acetone solution of
DDEt, containing a known amount of the compound, at 0. 01 ml per min,
into one of the mixing cells with a Sage syringe pump (Model 255-1).
Complete mixing of the toxicant with the incoming water was accom-
plished by employing a magnetic stirrer. Analysis of the DDEt solution
showed an average DDEt concentration of 0. 05 ppm.
Fifteen sexually immature minnows, selected from the stock procured
on January 20, 1971, were weighed and measured for length and placed
in each adult tank. They were weighed and measured again at the end
of the study. Oregon Moist Trout Pellets were provided ad libitum once
daily except on weekends. Water temperature was maintained at 23+. 1 C
throughout the test. The fluorescent lamps, controlled by an automatic
timer, were turned on at 6:00 a.m. each day and were turned off accord-
ing to the photoperiod schedule included in the appendix. Initial day length
was set for 13 hr, corresponding to April 1 on the schedule. Day length
was adjusted on the 1st and 15th of each month. Except on weekends,
daily observations on behavior were made and mortalities recorded.
When the males began to display sexual colors, three spawning tiles,
constructed of 4-in. half-sections of 6-in. diameter asbestos-concrete
drain pipe, were placed in each adult tank. The tiles were inspected for
eggs after 12 noon each day except on weekends. Eggs from each nest
were removed from the tiles and transferred to petri dishes for counting.
Since several females often deposit eggs in the same nest, the eggs were
examined for stage of development through a low-powered binocular
microscope. Eggs in distinctly different developmental stages were
counted as separate spawns.
Usually 50 unbroken eggs were selected from each non-weekend spawn
and incubated by the rocking egg cup method described by Mount (Refer-
ence 32). The egg cups were constructed of 3-in. sections of 2-in. diam-
eter, heavy walled polyethylene pipe with Nitex screening material (40
meshes per in. ) heat-sealed to one end. An electric motor was used to
gently oscillate the cups containing the eggs up and down in the test medium
3 times per min. The eggs were inspected daily and the dead eggs removed.
When the eggs began to hatch, they were left undisturbed until hatching was
complete.
The larvae were counted and 25 or more transferred to rearing chambers
and reared for 30 days. The larvae were fed a slurry of Oregon Moist
Trout pellets mashed finely in water ad libitum once a day, except on
The surviving larvae were counted and the caudal length
59
-------
recorded at the end of the 30-day test.
Data and Discussion
During the 28-week exposure period, the adult fish showed no signs of
distress. Very few adult fish died during the test. Percent survival
for controls was 97% or 29 of 30 fish. Percent survival for DDEt-
treated fish was 87% or 26 of 30 fish. Fish that died did not show
symptoms of the so-called "benzene bends" which have been observed
in minnows known to have died from DDEt-exposure (see later in this
section). There are insufficient data to indicate whether there is a
statistical difference in survival rate of the control group and the DDEt-
treated colony.
To evaluate the effects of DDEt on the growth of the adults, all fish were
weighed and measured for length at the beginning and at the end of the
study. The initial mean length, calculated by averaging the initial
lengths of fish in both treatment groups, was 55. 2 mm. This mean was
subtracted from the individual lengths measured at the end of the test.
The difference was considered the individual change in length. The mean
gain in length was 9. 5 mm for controls and 8. 0 mm for the DDEt-treated
fish. The difference in the mean gains was not significant by the Students
t-test (tca]^c = 1. 095; t Q^ = 2. 01). There was, however, an appreciable
difference in the gain in weight. The initial common mean was 2. 30 g.
The mean gain for controls was 1.46 grams and for the DDEt-treated
fish, 0. 92 g (tcalc = 7. 13; t. 05 = 1. 81).
Spawning commence~d on 13 May and continued until 5 August. The number
of spawns produced by each duplicate colony varied, but the pooled totals
were similar (Table 38). The controls spawned 23 times while the DDEt-
treated fish spawned 24 times. Exposure to DDEt also did not appreciably
affect the number of spawns produced per female. The average spawns
per female for controls was 1. 38; for the DDEt-treated colony, the aver-
age was 1. 32. Although the DDEt-treated colonies produced 140% more
eggs than the control colonies, the variation in the number of eggs pro-
duced per spawn was so great that there was no significant difference in
the mean number of eggs produced per spawn (tca^c = 0. 51; t^ 05 = 1. 68).
Exposure to DDEt also had no effect upon egg hatchability.
Data, from 30-day growth and survival tests, carried out on .larvae hatched
from eggs incubated under control conditions and in DDEt-saturated water,
indicated a deleterious effect of DDEt. Of 6 groups of "larvae reared in
DDEt-saturated water, 4 suffered total mortality within two weeks. Less
than half of the initial number of larvae in the remaining two groups sur-
vived the 30-day test. Survival of larvae in seven control groups ranged
from 38% to 98%. The average was 67%. Growth, as measured by ,
peduncle length, was not affected. The data are presented in Table 39.
60
-------
TABLE 38
REPRODUCTION DATA FOR FATHEAD MINNOWS
EXPOSED TO DDEt-LADEN WATER
Control DDEt-Treated Fish
Group: A B
No. of males 675 7
No. of females 9 8 10 8
No. of spawns 9 14 14 10
Spawns/female 1.0 1.8 1.4 1.2
Av. eggs/spawn 171 £ 60 228-^342 359 £ 354 157 ±.91
Total eggs produced 1540 3189 5029 1573
Percent hatch(N) 74(3) 83(11} 74(9} 75(5)
TABLE 39
THIRTY-DAY LARVAL GROWTH AND SURVIVAL
OF FATHEAD MINNOWS IN CONTROL
AND DDEt-SATURATED WATER
Control DDE t-treated
No. of test groups 7 6
Mean survival (%) 67 ± 23 15 +_ 23
Mean length 8. 9 mm 8. 6 mm
Six additional larval growth and survival tests were conducted on larvae
which had hatched from control eggs and were then exposed to water
saturated with DDEt. None of the larvae survived longer than 10 days.
The test data indicate that continuous exposure to DDEt-saturated water,
containing about 0. 05 ppm DDEt, does not affect behavior or survival
of adult fathead minnows. Although exposure to DDEt has no effect upon
growth as measured by increase in length, it does significantly reduce
the rate of weight gain by adult minnows. The minnows spawned normally
in DDEt-saturated water and the percent hatch of the eggs incubated in
such water did not differ appreciably from the hatching percentage of
control eggs. Although the larvae from eggs incubated in DDEt-saturated
water did not differ in appearance from control larvae, they survived
poorly in water containing DDEt. Since control larvae also survived
poorly in DDEt-saturated water, it is likely that DDEt affects the larvae
directly and that mortality among larvae exposed to DDEt is due only
slightly or not at all to effects of DDEt upon egg development or the adult
61
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reproductive system.
Larval Growth and Survival in Diluted
DDEt and TTTB -Saturated Water
Experimental Design
Since DDEt-saturated water proved to be toxic to fathead minnow larvae,
tests were undertaken to determine the maximum non-toxic level for
DDEt. Similar tests were conducted on dilutions of TTTB-saturated
water.
The tests were conducted in 1 x 1 x 2 ft (HWLi) tanks. A Mount-Brungs
chemical diluter was employed to dilute the saturated toxicant solutions
at concentration intervals of 50%. The toxicant was delivered to the
initial dilution cell of the diluter with a Harvard infusion pump equipped
with a 50 ml syringe containing an acetone solution of the toxicant. The
diluter was adjusted to deliver the toxicant solutions to the test tanks at
a rate providing five tank volume turnovers per day. The depth of the
test solution in each tank was 6 in., the volume was approximately 28j£ .
The larvae used in these tests were hatched from eggs produced by
stock minnows. During the test, the larvae were housed in specially
constructed chambers which could be removed from the test tanks for
cleaning or whenever a fish census was needed. The chambers were
constructed of heavy-walled acrylic tubing with an inside diameter of
4 in. and a length of 7. 5 in. Two 2x5 in. windows, covered with Nitex
screen (40 meshes per inch), were installed around the circumference of
the chamber, 1. 5 in. from the closed bottom. The test solution from the
diluter was delivered to double-drain mixing cells, one located above each
chamber. Half of the delivered volume entered directly into the chamber
while the other half was delivered to the outer test tank. The larvae were
fed on Oregon Moist Trout pellet slurry, ad libitum, once a day except on
weekends. The length of each test was 30 days. Two tests were conducted
on DDEt. TTTB was tested only once.
Data and Discussion
In the first DDEt test, 50 larvae were placed in each chamber. By the
end of the test, all fish exposed to the DDEt-saturated solution (50 ppb)
had died. Only 4% of the fish exposed to 25 ppb DDEt survived. Survival
at DDEt concentrations up to 12 ppb was similar to survival among con-
trols (Table 40). The growth data were examined, but no conclusions
could be reached. The mean length of the survivors exposed to 25 and 6
ppb DDEt was slightly lower than the mean lengths of survivors in the
other colonies; however, the effect may have been due to space and food
limitations as these were the largest colonies. The data from this test
indicate a minimum non-toxic DDEt concentration of 12 ppb with the
maximum acceptable dose somewhere between 12 and 25 ppb.
62
-------
TABLE 40
THIRTY-DAY GROWTH AND SURVIVAL OF CONTROL
LARVAE EXPOSED TO VARIOUS CONCENTRATIONS OF
DDEt-LADEN WATER (Test 1)
Length, mm
Estimated
Fraction % Standard
DDEt (ppb) Surviving Survival Mean Deviation
50 0/50 0
25 2/50 4 8.75 0.35
12 31/50 62 8.62 1.21
6 20/50 40 9.58 0.89
3 32/50 64 8.91 1.18
Control 25/50 50 9.73 1.10
The second test on DDEt resulted in slightly different data (Table 41).
The data indicate that the MATC of DDEt may be lower than 6 ppb.
This compares with an MATC estimate of 12 to 25 ppb based on the
data from the first test. Although the mean length of the larvae
increased with decreasing concentration, the differences were not
significant. A comparison of the mean length for fish reared in 1/2
saturated water (25 ppb) and controls gave a calculated "t" value of
1.43, whereas t^ 05 = !• 74.
TABLE 41
THIRTY-DAY GROWTH AND SURVIVAL OF CONTROL
LARVAE EXPOSED TO VARIOUS CONCENTRATIONS OF
DDEt-LADEN WATER (Test 2)
Mean Peduncle Length
Fraction % and Estimated Standard
DDEt (ppb) Surviving Survival Deviation (mm)
50 0/25 0
25 6/25 24 7.6^1.2
12 6/25 24 7. 9 ±1.2
6 10/25 40 8.0 J: 1.3
Control 14/25 56 8.5 ±. 1. 7
63
-------
Fathead minnow larvae, exposed to TTTB solutions of concentrations
up to saturation for 30 days, did not appear affected with respect to
survival or growth (Table 42).
TABLE 42
THIRTY-DAY GROWTH AND SURVIVAL OF FATHEAD MINNOW
LARVAE EXPOSED TO DILUTED TTTB-SATURATED WATER
Fraction % _ Length (mm) _
TTTB (ppb) Surviving Survival Mean Standard Deviation
25/25 100 9.4 0.9
^0.5 24/25 96 8.8 1.1
^0.2 17/25 68 8.2 1.2
^0.1 24/25 96 9.0 0.9
^0.06 16/25 64 9.0 0.7
Control 25/25 100 8.7 1.2
In summary, the maximum acceptable DDEt concentration for fathead
minnow larvae, based on 30-day larval growth and survival tests, is
less than 1/8 the concentration of a saturated aqueous solution of
DDEt. This concentration is approximately 6 ppb. This is the lowest
concentration which produced a measurable effect on any life stage of
the fathead minnow. TTTB saturated water ( < 1 ppb) has no apparent
effect upon growth or survival of fathead minnow larvae.
Chronic Studies with DDEt-Laden Food
Experimental Design
The initial selection of the range of test concentrations for use in a
chronic bioassay, where one of the major parameters to be measured
is productivity, is often difficult. Ideally, the test concentrations or
dosages selected for any bioassay should cover a range that includes
toxicant levels which will produce the desired effect or effects. The
magnitude of effect should increase in proportion to concentration.
Selection of a concentration range for a chronic bioassay can often be:
made by carefully examining the dose-response curve constructed from ;
data obtained from acute bioassays performed on the toxicant in question.
Generally, the TLm, the slope of the curve and the concentrations which
produce no measurable effect are taken into consideration.
64
-------
Acute toxicity tests performed prior to the chronic tests conducted
on DDEt saturated water indicated that DDEt is relatively non-toxic.
Although the 96-hr TLm could not be determined, the data indicated
that it would probably exceed 6 ppm and that a concentration of
0. 10 ppm would probably be a safe concentration for all life stages
of the test fish. This early assumption, although erroneous, led
to the belief that the maximum acceptable DDEt concentration would
exceed saturation, or 0. 05 ppm.
For the chronic exposure tests, DDEt concentrations of 5, 1, 0. 2 and
0. 07 ppm were initially selected. The lowest concentration was thought
to be the concentration of DDEt saturated water. This was later revised
to 0. 05 ppm. Agrimul 70-A was used as a dispersant for DDEt and
employed only in the 5, 1 and 0. 2 ppm tests. Water containing Agrimul
only was used in the control tests. A chemical diluter (Mount and
Brungs, Reference 23) was used to dilute and deliver the DDEt-Agrimul
suspensions. The DDEt-saturated water did not contain Agrimul and
the control medium was in unadulterated laboratory water.
Analysis of the Agrimul suspensions showed only trace amounts of
DDEt. Addition of a recirculating pump to stir the Agrimul-DDEt
stock solution and increasing the amount of DDEt in the stock solution
did not improve the situation. The DDEt saturated water was found to
contain 0. 05 ppm DDEt consistently.
These problems led to abandoning of the tests involving Agrimul, and
the development of another method for testing DDEt levels exceeding
saturation. The method of choice was the administration of DDEt to
the minnows via their diet. The procedures used in these feeding tests
are described below.
The chronic feeding experiments were preceded by a series of acute
tests conducted in 1 x 1 x 1 ft. tanks containing 6 in. of water (approxi-
mately 14 1). The water in these tanks was changed at a rate of 200 ml
per minute or 10 tank volumes per day. For this test, 1, 1. 8, 3. 2,
5. 6 and 10 grams of DDEt in 10 ml of acetone were added to 10 g of
Oregon Moist Trout pellets and the solvent evaporated off at room
temperature. Food for the control colony was treated in the same
manner with solvent only. Analysis of the treated food showed the
DDEt content to be within 2% of the calculated level.
The fathead minnows selected for testing were fasted for 24 hours and
weighed in groups of 10 before being placed as a group into the test
tanks. The DDEt-laden food was provided once daily, except on week-
ends, at a rate of 5% of body weight. The tests were terminated after
10 days. The dosages and results are shown in Table 43.
65
-------
TABLE 43
ACUTE TOXICITY OF DDEt TO FATHEAD MINNOWS
FED DDEt-LADEN FOOD FOR 10 DAYS
Treatment Mg DDEt/gm % DDEt Appearance
Group fish/day in diet of fish
Control 0 0 Normal
1 4.6 9.1 Normal
2 7.6 15.2 Normal
3 12. 1 24.3 Emaciated
4 17.8 35.8 Emaciated
5 25.0 50.0 Emaciated
All but one of the test fish survived the 10 day test. However, although
the fish readily consumed the treated food initially, those receiving
diets containing 24. 3% or more DDEt began to eat less food after
three days. Fish in these groups were noticeably emaciated by the
seventh day and one fish in the colony receiving the 24. 3% diet died.
Anorexia and emaciation became more and more evident with time.
Fish receiving less than 24. 3% DDEt in their diet readily consumed
food throughout the test and did not appear affected. On the basis of
this test, the maximum dosage selected for the chronic test was 2 g
DDEt per 100 g of food or approximately a 2% diet (i. e., ca 10% of
dose giving an effect in seven days or less).
The long term tests were initiated on June 16, 1971. Test procedures
and conditions were the same as in the long term study on DDEt saturated
water with a few exceptions. Instead of delivering the test water to the
larval tanks via mixing cells, the water was fed directly into each '
larval tank by means of individual feedlines. As before, the water
passed through the larval tanks and into the adult tanks before being
sewered.
The initial day length was set at 13. 5 hrs, corresponding to mean day
length for April 1 on the attached photoperiod schedule (Appendix).
After the maximum day length on the schedule was reached, it was
further increased to 16 hrs and maintained at this level for the remainder
of the study.
The test fish were selected from the minnow stock purchased on May 18,
1971. The fish were individually measured for length (fork lengths) and
then weighed in groups of five. The groups were assigned to the test
tanks by random stratified assortment until each tank contained 15 fish.
The 10 tanks for the adult fish were than randomly assigned numbers
and placed on the tank racks serially to minimize possible effects
caused by tank location.
66
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The DDEt-laden food was prepar ed by adding 0. 002, 0. 02, 0. 20 and
2. 0 g of DDEt in 100 ml of acetone to 100 g of Oregon Moist Trout
pellets and evaporating the solvent at room temperature. Food for
the control colonies was prepared in the same manner with the same
amount of solvent added. The food was kept frozen, but thawed each
day before use.
The fish were fed once daily, except on weekends, at a rate of 5%
of body weight. To establish the food-weight relationship, the fish
in each tank were weighed as a group every two weeks. The dosages
were 0, 0.001, 0.01, 0. 10 and 0. 98 mg DDEt per g fish per day.
These dosages are equivalent to 0. 02, 0. 2, 2. 0 and 20 mg DDEt per g
food per day. Food left uneaten after 4 hr was siphoned out and
discarded. When the fish began to spawn, the weighing schedule was
abandoned so as not to disturb the fish and food was provided once a
day ad libitum.
When the fish reached sexual maturity and could be identified with
respect to sex, a screened partition was installed in each tank so
that the volume of one section was twice the other. All females and
three males were permitted to remain in the larger section and were
provided with three spawning tiles. The rest of the males were placed
in the smaller section. Males with the females were replaced whenever
any of these males died.
Data and Discussion
During the first 10 weeks all adult fish receiving 20 mg DDEt per g
of food developed symptoms of a syndrome known as the "benzene bends"
and died. The symptoms started with refusal of food, followed by
progressive emaciation. In the later stages, the fish developed dorso-
ventral spinal curvature and often swam erratically near the bottom of
the tank at an angle varying from 45 to almost 90 degrees from horizontal.
The terminal stage was marked by extreme emaciation and feebleness.
At this stage the fish lay on its side on the bottom of the tank. Except
for opercular nr> vement, the fish generally lay motionless until death.
Figure 2 compares the appearance of a typically affected fish with that
of an unaffected fish.
Mortality among fish in the remaining colonies varied considerably
between duplicate tests (Table 44) and appeared unrelated to DDEt
dosage. Although typical toxic symptoms developed in some of the
fish, the occurrence of these symptoms was not consistent. The two
dead fish recorded for group 1 (0. 02 mg DDEt} and all six dead fish
from, both of the groups which received a dosage of 0. 2 mg DDEt per g
food per day developed definite toxic symptoms. These fish represented
only 25. 8% of the total number of fish dying during the 28- week test.
Interestingly, of the 31 dead fish recorded (excluding those which
had received the highest dose) 4% were males. In terms of sex, 1%
of the remaining males and only 7% of the remaining females died.
Low survival among males was also observed in similar tests with
TTTB laden food.
67
-------
.
Control (A)
DDEt -Exposed (B)
A
Fish A - Control
Fork Length 62mm
Weight 3.33g
Fish B - DDEt -Exposed
Fed 980mg DDEt/kg Body Weight/Day for 50 Days
Fork Length 55mm
Weight 1.51g
Figure 2. EFFECT OF FEEDING HIGH DOSE RATE OF DDET
ON FATHEAD MINNOW
68
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TABLE 44
MORTALITY OF FATHEAD MINNOWS
FED DIFFERENT DOSAGES OF DDEt IN THEIR DIET
Females
Males
Total
Mg DDEt/gm Food Group Initial Final Dead Initial Final Dead Dead(%)
Control
Control
0.02
0.02
0.20
0.20
2.0
2.0
20.0
20.0
1
2
1
2
1
2
1
2
1
2
13
11
12
12
12
10
11
13
11
12
6
10
10
10
11
6
10
3
11
12
7
1
2
2
2
4
1
10
11
12
2
4
3
3
3
5
4
2
4
3
2
3
3
2
3
5
4
2
0
0
0
1
0
1
0
0
0
0
4
4
42
14
14
20
33
27
7
7
100
100
The reason for low male survival is not known. The amount of DDEt in
their diet did not appear responsible. One explanation is that the
population may have contained a large percentage of second season
males. According to Markus (Reference 33), 80 to 85% of the first
year spawners die after spawning. The carryovers almost invariably
die after spawning the second time. The test fish for this study and
the study on TTTB laden food were selected from the minnow stock
acquired in May. Why the May-acquired stock contained 78% males
and the January stock contained only 48% males is beyond the scope
of this study.
During the first 12 weeks before spawning commenced, the control
colonies gained an average of 0. 26 g per fish per two week interval.
This average was calculated from pooled data from both duplicate
colonies.
Colonies which received 0. 02, 0. 20 and 2. 0 mg DDEt diets gained
weight at an average rate of 0. 25, 0. 26 and 0. 21 g per fish. Fish
fed 20 mg DDEt per g food were weighed twice a month for only
eight weeks. The bi-monthly growth rate was only 0. 017 g.
In general, the total gain in weight was somewhat similar for all
groups except those on the 20 mg DDEt diet and possible those on the
2. 0 mg DDEt diet (Table 45). The latter gained only 74% as much as
controls and the former gained only 3. 4% as much as controls. In
all groups except those en the 20 mg DDEt diet, the mean gain in
length was similar.
69
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TABLE 45
GROWTH OF FATHEAD MINNOWS FED DDEt-
LADEN FOOD FOR 28 WEEKS
Mean individual fork
length (mm)
Mean individual weight
(g)
Control
Control
0.02
0.02
0.20
0.20
2.00
2.00
20.0
20.0
* 8 weeks
1
2
1
2
1
2
1
2
1
2
Initial
54.8
53.1
51.5
51.2
55.1
52.9
55.3
51.9
52.8
54.6
Final AJLength
67.8
65.8
66.1
65.0
68.5
65.6
67.8
65.8
-
—
13.0
12.7
14.6
13.8
13.4
12.7
12.5
13.9
-
_
Initial
2.2
1.9
1.8
1.7
2.1
1.7
2.2
1.7
1.9
2.1
Final ^Weight
5.1
4.4
4.2
4.3
5.4
4.2
4.1
3.9
2.0
2.2
2.9
2.5
2.4
2.6
3.3
2.5
1.9
2.2
0. 1*
0.1*
The minnows started to spawn on September 13 and continued until
January 19, a period of about four months. The sex ratio and data
on spawning and egg hatching success for the 10 colonies are listed
in Table 46. Except for the two colonies which were fed 20 mg DDEt
per g food, there was no consistent relationship between the amount
of DDEt in the diet and the number of spawns per female. Although
one of the control colonies produced 7.5 spawns per female, its
duplicate produced only 0.75 spawns per female, a performance not
appreciably different from that of any other colony from which spawns
were obtained.
Hatching success of the eggs produced by colonies fed 2 mg DDEt
per g of food was significantly lower than the hatching success of eggs
produced by controls. A Students "t" test m the difference between
the mean percent hatch based upon pooled data, gave at , of 2. 53,
whereas tQ Q5 is 1.77. Hatching success of the two other"colonies
which produced eggs was similar to that of controls.
The fry from all colonies normally hatched within five to seven days
after they were transferred to the incubation cups. There was no
noticeable difference in the appearance of the fry upon hatching.
Hatching percentages varied considerably and there was no appreciable
difference in the mean length of the fish after 30 days (Table 47). Due
to the paucity of data, the effect of DDEt on larval growth and survival
cannot be evaluated.
70
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TABLE 46
Group:
No. of males
No. of females
No. of spawns
Spawns /female
Av. eggs /spawn
Eggs produced
Total
Percent hatch (N)
EGG PRODUCTION AND HATCHABILJTY
BY FATHEAD MINNOWS FED DDEt- LADEN FOOD
mg DDEt per g
1
3
2
15
7.
119
Control
2
4
3
5 0.75
.9 108.7
1799 326
85.
8(8)90.0(3)
0.02
1 2
3 3
3 3
4 0
1.33 0
157.0 0
628 0
96.0(3) -
0.
1
3
3
1
0.33
393
393
_
20
2
3
5
2
0.40
170.0
340
92.0(3)
Food
1
3
4
0
0
0
0
-
2.0
2 1
3 0
2 0
4 0
2.0 0
67.8 0
271 0
76.6(3) 0
20
2
0
0
0
0
0
0
0
-------
TABLE 47
PERCENT SURVIVAL AND MEAN LENGTH OF FATHEAD MINNOW
LARVAE FED DDEt-LADEN FOOD
Test
Number
1
2
3
4
5
6
7
8
Mean Survival, %
Mean length, mm
0
24.0
2.9
45.7
30.2
40.9
14.9
40.0
17.5
27.0
9.1
mg DDEt
0. OZ
0
42.0
14.0
18.7
8.9
per g food
0.20 2.0 20.0
39.0 13.7 0
32.0 0 0
0
23.3 6.8 0
10.0 9.6
Administered per os, DDEt appears considerably less toxic to fish than
DDT administeredTin the same manner. Cutthroat trout, fed a pelleted
diet containing DDT at a rate of 1 mg DDT per kg every seven days showed
a significantly high mortality rate compared to controls after four months
of feeding (Reference 34). The calculated daily rate of DDT intake would
be 0. 183 mg per kg (1 mg/seven days). In the present study, fathead
minnows on the 0. 02 mg DDEt per g food were fed at a rate of 10 mg per
kg body weight per day during the first three months and possibly more
subsequently when they were fed ad libitum. At this dosage rate, the
minnows were not affected with respect to growth survival and egg
production. The 2. 0 mg DDEt per g food diet, fed at a rate of at least
100 mg DDEt per kilogram body weight per day, reduced egg hatchability
significantly in fathead minnows and probably reduced growth somewhat.
Neither dosages affected survival.
Species differences could account for some of the difference in toxicity;
however, it is unlikely that the 50-500-fold greater toxicity of DDT
could be due to species differences alone. The 96-hr TLm of DDT to
fathead minnows, goldfish, guppies and bluegills range only from 0. 021
to 0. 056 ppm (Reference 27), and the 96 hr TLm for rainbow trout range
from 0. 024 to 0. 074 ppm (Reference 35). For these five fish species
the highest TLm concentration is no more than four times the lowest.
72
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Chronic Study on TTTB-Laden Food
Experimental Design
The procedures employed in this study were the same as those used
in the chronic study on DDEt-laden food. The test tanks, however,
were 1 x 1 x 3 ft (HWL) tanks with two sections 12 x 10 x 6 in.
partitioned off with 40 mesh Nitex screen to serve as larval rearing
areas. These tanks permitted the adults and larvae to be maintained
in one tank and conserved a considerable amount of laboratory space.
Since TTTB is only sparingly soluble in acetone, the treated food was
prepared by dissolving 0. 002, 0. 02, 0. 20 and 2. 0 g of TTTB in 100 ml
of tetrahydrofuran (THF), mixing the solution with 100 g of Oregon
Moist Trout pellets and evaporating the solvent at room temperature.
Preliminary tests showed that 555 mg THF per liter was not toxic to
fathead minnows over a period of 96 hours and that food containing
as much as 24% TTTB had no effect upon fathead minnows over a
period of 10 days.
The study was initiated on June 16, 1971 with minnows purchased on
May 18, 1971. The initial number of fish per duplicate tank was 15.
Data and Discussion
During the 28 week test, mortality was similar for all treatment
groups (Table 48). A total of 48 fish died during the test; 94% of
these were males. The male population suffered 39% mortality
and the female population only 9%. None of the dead fish showed
symptoms of the "benzene bends. " Of the 48 total, 23 or about 48%
died from an apparent bacterial infection. The causative agent was
investigated but not identified. Tetracycline treatment for 3-4 days
was effective in controlling the infection. Subsequent deaths did not
appear dose-dependent.
TABLE 48
MORTALITY OF FATHEAD MINNOWS FED DIFFERENT
DOSAGES OF TTTB IN THEIR DIET
mgTTTB/ ______________ T<>tal
g food Group Initial Final Dead Initial Final Dead Dead(%)
Control 1 11 744 3 1 33
Control 2 13 3 10 2 2 0 68
0.02 1 11 744 4 0 28
0.02 2 11 4 7 4 3 1 53
0.20 1 13 9 4 2 2 0 27
0.20 2 12 12 0 3 30 0
2.0 1 13 8 5 2 2 0 33
2.0 2 13 7 6 2 2 0 40
20.0 1 10 7 3 5 4 1 27
20.0 2 9726 6 0 14
73
-------
The fish began to spawn on August 18. Up to this date the fish were
weighed bi-monthly. The mean increase in individual weight,
calculated from pooled data was 0.29, 0. 21, 0.25, 0. 26 and 0. 25 g,
respectively, for controls and for colonies receiving 0.02, 0.2, 2.0
and 20 mg TTTB per g food per day. The slightly higher growth rate
of controls during this period was not carried through to the end of
the test. Fish in all colonies showed similar overall lengthand weight
gains (Table 49).
TABLE 49
GROWTH OF FATHEAD MINNOWS FED TTTB-LADEN FOOD
FOR 28 WEEKS
mg TTTB/ Mean fork length (mm)
Mean individual weight (g)
g food
Control
Control
0.02
0.02
0.20
0.20
2.0
2.0
20.0
20.0
Group
1
2
1
2
1
2
1
2
1
2
Initial
52.2
53.9
53.5
53.9
53.1
54.0
54.1
54.5
56.1
55.1
Final A
65.0
65.2
65.9
66.6
66.9
66.9
67.5
70.9
66.7
67.4
Length
12.8
11.3
12.4
12.7
13.8
12.9
13.4
16.4
10.6
12.3
Initial
1.7
1.7
1.8
1.8
1.7
2.0
1.9
1.9
1.9
2.1
Final
4.1
4.7
3.9
4.5
4.4
4.5
4.6
5.3
4.3
4.3
AWeight
2.4
3.0
2.1
2.7
2.7
2.5
2.7
3.4
2.4
2.2
The number of spawns per female was inconsistent. The two colonies
on the 2 mg DDEt diet spawned a total of 12 times for spawn per female
records of 4 and 2. One control colony spawned three times per female
but all other colonies spawned no more than 0. 5 times per female
(Table 50).
74
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TABLE 50
-j
Ui
No. of males
No. of females
No. of spawns
Spawns /female
Mean eggs/spawn
Total eggs
Percent hatch (N)
'RODUCTION AND HATCHABILITY BY FATHEAD MINNOWS
FED TTTB-LADEN FOOD
mg
Control
1
3
4
1
0.25
40.0
40
25(1)
2
3
2
6
3.0
321.0
1926
71.6(6)
0.
1
3
4
2
0.50
14.5
29
100(2)
02
2
3
4
1
0.25
30.0
30
98(1)
1
3
2
0
0
0
0
_
TTTB per
0.2
2
3
1
0.33
83
83
48(1)
g food
2 .0
1
2
8
4.0
148.0
1184
63.8(8)
2
3
2
4
2.0
90.2
361
85(4)
1
3
5
0
0
0
0
-
20
2
3
6
1
0.17
22
22
90(1)
-------
Hatching percentages also varied considerably (Table 50). In general,
the hatching percentage of eggs from fish fed TTTB-laden food was no
worse than the hatching percentage of eggs from controls.
The data on growth and survival of larvae fed the same diet as the
parental fish are shown in Table 51. Very little data was obtained
on this phase of testing as none of the treatment groups produced a
sufficient number of spawns. The data obtained were highly variable.
None of the individuals in five of the larval groups tested at 0. 2 mg
TTTB/g food and higher survived the 30 day exposure period. While
this may indicate a possible effect of TTTB, one of the groups tested
at 2.0 mg TTTB/g food showed 84.4% survival which was higher than
that of any other group in the entire test. Also, survival of groups
from tank 2, 2. 0 mg TTTB/g food, compared favorably with survival
of control groups. Hence, the effect of TTTB on the survival of
fathead minnow larvae is questionable; the tests bear repeating.
In summary, the data indicate that a diet of Oregon Moist Trout pellets
containing up to 2 g DDEt per 100 g of pellets has no effect upon the
growth, survival, or egg production of fathead minnows. Data on egg
hatchability and larval growth and survival was insufficient for
statistical treatment; hence, no conclusions can be made on the effects
of TTTB-laden food on these parameters.
TABLE 51
THIRTY-DAY GROWTH AND SURVIVAL OF
FATHEAD MINNOW LARVAE FED TTTB-LADEN FOOD, %
mg TTTB per g food
Control
0.02
0.20
Group: 1
No 24.0 28.0 16.0 No 0
spawn spawn
44.4 36.4
26.0
46.0
42.9
2.0
20.0
T
0
0
0
4.0
84.4
18.0 No 0
spawn
36.2
28.4
Mean
survival, % 37 32 16
Mean
length, mm 9.0 8.4 8.8
18 28
8.7 9.0
76
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SECTION VII
ANALYSIS OF STUDIES
The studies described in the preceding sections represent the initial
development of a concept of controlled destruction of field-applied
pesticides, such as DDT. The feasibility of the overall concept was
shown in earlier studies (Reference 1). Two areas were analyzed:
the basic chemical reactions involved in degrading DDT, and the
expected safety of the products to various life forms.
Both copper-catalyzed aluminum and copper-catalyzed iron reductants
appear to give sufficiently rapid and extensive degradation of DDT to be
useful for field degradation. The decomposition with the Al'Cu system
requires 24 hr at 25°C, and 4 hrs at 40° (simulating summertime
conditions in warmer climates), while 1-2 weeks at 25°C and 8 hrs at
40 C is needed for decomposition by the Fe* Cu system. Although
neither system gives as rapid decomposition as the copper-catalyzed
zinc reductant, the systems are sufficiently rapid for destruction of
field-applied DDT.
The use of copper-catalyzed aluminum or iron overcomes a potential
problem attendant to the use of catalyzed zinc reductant. The reaction
involving zinc produces 3 equivalents of zinc ion per equivalent of DDT
reduced. This is a drawback since zinc ion in sufficient quantity is
known to be toxic to fish. Sources cited in Reference 1 indicate that
zinc ion concentrations as low as 0. 01 mg/1 may cause deleterious
effects to certain fish species; with other fish, the median tolerance
limit may be as high as 35 mg/1 of zinc ion. However, the use of
catalyzed iron or aluminum appears to obviate this problem, since
the iron or aluminum ions in reasonable quantities are apparently not
toxic to fish. Importantly too, only one equivalent of iron or aluminum
is required theoretically per equivalent of DDT, so that the amount of
dissolved metal is reduced significantly. The economic advantage of
reduced metal consumptioni s also important. The comparison of
theoretical metal usage and cost is given in Table 52.
77
-------
TABLE 52
THEORETICAL METAL USAGE AND REDUCTANT COST
FOR REDUCTIVE DEGRADATION OF DDT
Al'Cu Fe'Cu Zn'Cu
Reductant Couple Couple Couple
Theoretical: 113
equiv. reductant/mol DDT
Theoretical: 0.025 0.052 0.28
Ib metal ion/lb DDT
Theoretical Reductant 1.0 0.2 5.5
cost* cents/lb DDT
* Based on Zn dust at $0.20/lb, Al powder at
$0. 40/lb, Fe powder at $0. 04/lb
The practical deployment of a reductively degradable form of DDT
apparently requires a source of acidity for the reaction. Requirements
for the avoidance of phytotoxic damage as well as economy of reagents
suggests that the amount of acid employed should be minimal. The use
of an acid which exists in solid state, and which might be placed in close
proximity to the reactants (DDT and reductant) appears to be a suitable
means for carrying out the reaction.
The discovery that moderately strong acids, such as sulfamic, are
preferred is important to the development of a practical system. The
difficulties in removing water of crystallization and in an early reaction
of aqueous acid and reductant appears readily solvable with proper
choice of non-aqueous solvent, or in using a technique such as spray
drying for application of the acid. Minimum acid requirements for
effective reaction have not been established, but the cost appears to
be low. The quantity of acid employed in early tests was 15 millimoles
of acid per gram of DDT. The cost of this quantity of sulfamic acid
would be about $0. 21 per pound of DDT; again, it should be emphasised
that minimum amounts of acid for effective action have not been determined
and it is expected that the quantity and cost can be appreciably reduced.
The dissemination of 1. 5 pounds of acid/acre (assuming 1 Ib/acre of DDT
and 1.5 Ibs sulfamic acid/lb DDT or 15 millimoles/g DDT) would appear
to offer no significant problem with respect to modifying soil characteristics,
The results involving tests of the integrated particle concept in which
a fine reductant particle was overlaid with the solid acid and the whole
covered with DDT, leads strongly to the conclusion that the concept is
both practical and workable. Only moisture is required to initiate the
78
-------
reaction. The inclusion of a slowly dissolving, permeating, eroding,
or rupturing membrane between the acid coat and the pesticide layer,
to provide the requisite delay to allow for pest control action, would
result in the desired controlled, self-destructing form of the pesticide.
Although the reported tests involved the catalyzed zinc reductant system,
all evidence points to equivalent degradation employing the catalyzed
aluminum and iron systems.
It would be important in considering this concept to examine the
properties of the integrated particle on dissemination. It has been
shown in an earlier analysis that 5-20/am particle size in aerosol
spray appears desirable on the basis of experience, as well as aero-
dynamic capture, settling velocity, and related factors (Reference 1).
While it may be considered that the pest control effectiveness of the
integrated particle is not affected by the process of forming the particle,
since the DDT is on the outside of the particle in unmodified form, close
examination of the concept is warranted. Important to the analysis is
the consideration of the mean interparticle distance in uniformly
dispersed particles, since too great a distance would reduce the
probability of effective pest control action. Calculations can then
show the number of particles per unit area, and the mean interparticle
distances, assuming uniform distribution. This calculation (Table 52)
is based upon the use of 4 Ibs of reductant and 1 Ib of DDT per acre.
TABLE 53
CALCULATED EFFECT OF PARTICLE SIZE ON THE
MEAN PARTICLE-PARTICLE DISTANCE
FOR Al« Cu AND Fe' Cu REDUCTANTS,
INTEGRATED SELF-DESTRUCTING PESTICIDE CONCEPT
Reductant Al'Cu Fe* Cu
Mean Particle- Mean Particle-
Particle Size -> Particle Dist. 2 Particle Dist.
yum particles/cm /u.m Particle/cm
5 2.5xl05 20 8.9xl04 33
10 3.2xl04 56 l.lxlO4 95
20 4.0xl03 160 1.4xl03 450
These distances appear sufficiently small so that minimal movement
by the pest will assure contact with the pesticide. Hence, pest control
action should be highly effective. As shown previously, particles of
5-20 >Um mean diameter should be readily handled by conventional
spray or dusting equipment.
79
-------
The fate of the degradation products in the environment also requires
consideration.
The copper-catalyzed aluminum reduction of DDT leads to the production
of TTTB and DDA as products, while the catalyzed iron reduction leads
mainly to the formation of TTTB. The product TTTB is insoluble in
water (1 ppb or less solubility at 40 C), and the solubility in the fat
triolein is 50-fold less than DDT. Hence, the material tends to
remain in the environment as an inert solid, rather than being
dispersed in land runoff, or in being transmitted in life forms through
fat-solubility, as DDT. Limited hydrolytic stability tests indicated
an intrinsic stability of the material, and other tests indicated
resistance to further chemical reaction. Attempts to measure the
vapor pressure were not successful, but the fact that the material is
a high melting solid (268-270 C melting point) suggests that the vapor
pressure is very low and evaporative losses would be negligible.
The product DDEt, obtained from the catalyzed zinc reduction of DDT,
is readily dispersed, however. The solubility in water is about 20-^fold
greater than DDT, and the solubility in the fat triolein is about 2-1/2-fold
greater than DDT. The vapor pressure of DDEt is about 80-fold greater
than DDT, and evaporative loss of the product from the field was
calculated to be significant. Indeed, long term laboratory experiments
of particle degradation have frequently led to lower than expected product
recovery; DDEt evaporationis believed responsible for the apparently
low recovery of the product.
The toxicity studies also showed a significant difference between the
two basic reductive degradation techniques; Al'Cu or Fe« Cu reduction
leading to TTTB (and DDA), or Zn« Cu reduction producing mainly DDEt.
No adverse effects were noted with TTTB, on the basis of long term
chronic effects, or 96 hr acute toxicity tests with fathead minnows.
The growth and survival of adults over a 7 month period resembled
control data. The egg production rate, hatchability and growth and
survival of freshly-hatched fry were all statistically similar to those
of unexposed control fathead minnow colonies. Feed tests in which
about 0. 1% of the diet consisted of TTTB (980 mg/kg body weight/day)
also produced no evident toxic effects.
Acute testing with DDEt, in which fathead minnows were exposed to
suspensions containing initially as much as 100 ppm DDEt, as well as
tests with saturated solutions (0. 05 ppm), produced no toxic effects.
Similarly, 7 month tests of the growth and survival of adult fathead
minnows, egg production, and hatchability appeared similar for control
groups and colonies exposed to DDEt-saturated water. However, a
significant mortality of freshly hatched fry exposed to DDEt-saturated
water was noted, and the concentration of DDEt had to be reduced to
about 6 ppb before fry mortality ceased. Thus, the maximum acceptable
toxicant concentration appears to be about 6 ppb. Tests in which DDEt
was added to the diet indicated that levels of DDEt greater than 10 mg/kg
body weight/day led to toxic effects. At high concentrations of DDEt,
80
-------
emaciation and dor so- ventral spinal curvature was noted. Grossly
exposed fish were observed to swim erractically before succumbing.
The symptoms are understood to be similar to the "benzene bends"
described for fish exposed to the chlorinated pesticide lindane.
In summary, a number of advantages of the catalyzed aluminum or
catalyzed iron reductant systems are shown over the catalyzed zinc
system. Foremost among these is the lack of any discernible toxic
effect on fish from either short term acute testing or long term chronic
testing. The reaction appears to proceed smoothly at reasonable rates
to a product shown to be inert to the environment under the conditions
tested.
The data indicate the reaction can be carried out in a practical way,
and means are suggested for the applicationcf the concept. The results
of this initial developmental study are believed to strongly fortify the
conclusions of the feasibility study that the technique is a practical and
useful one. Further, development and implementation of the concept
is strongly recommended.
81
-------
SECTION VIII
ACKNOWLEDGEMENTS
This project was funded by the United States Environmental
Protection Agency, Contract 14-12-922 to the Environgenics
Systems Co., El Monte, California.
The support of the project by the Environmental Protection
Agency and the help provided by Dr. H. P. Nicholson, Project
Officer and Dr. Robert Swank (Southeast Environmental
Research Laboratory), is acknowledged with sincere thanks.
The help of Mr. John Eston of the National Water Quality
Laboratory of the Environmental Protection Agency, Duluth,
Minnesota, has been appreciated. His suggestions relative
to the dispersal of toxicants, and in the biology and rearing of
fathead minnows have been very helpful.
The assistance of California Fish and Game biologists in
enabling the rearing of disease-free fish is also acknowledged.
The contributions of Mr. Harold Wolf, Fish and Game Patho-
logist, and Mr. William Richardson, Inland Fisheries Section,
are particularly noted.
83
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SECTION IX
REFERENCES
1. Investigation of Means for Controlled Self-Destruction of Pesticides,
Aerojet Final Report on FWQA Contract 14-12-596, June 1970.
Water Pollution Control Research Series 16040 ELD 06/70.
2. Mosier, A. R. , Guenzi, W. D. , and Miller, L. L. , "Photochemical
Decomposition of DDT by a Free-Radical Mechanism, " Science,
164, 1083-1085 (1969).
3. Menzie, C. M. , "Metabolism of Pesticides, " U. S. Dept. of
Interior, Bureau of Sport Fisheries and Wildlife, Washington,
D. C. , Special Scientific Report - Wild Life No. 127, July 1969.
4. Bowman, M. C. , Schechter, M. S. , and Carter, R. L. , "Behavior
of Chlorinated Insecticides in a Broad Spectrum of Soil Types, "
J. Agr. Food Chem. , 13, 360-365 (1965).
5. Washburn, E. W. , ed. , International Critical Tables, McGraw Hill,
New York, 1927, Vol. II, p. 435.
6. Thomson, S. J. and Webb, G. Heterogeneous Catalysis, Wiley,
New York, 1968.
7. Grummitt, O. , Buck, A., and Jenkins, A., "1, l-Di(p-chlorophenyl)-
1, 2, 2, 2-tetrachloroethane, " J. Amer. Chem. Soc. , 67, 155-156
(1945).
8. Biggar, J. W. , Dutt, G. R. , and Riggs, R. L. , "Predicting and
Measuring the Solubility of p, p'-DDT in Water, " Bull. Environ
Contamination and Toxicology, 2, 90-100 (1967).
9. Gunther, F. A. , Westlake, W. E., and Jaglan, P. S., "Reported
Solubilities of 738 Pesticide Chemicals in Water, " Residue Rev. ,
20, 1-148 (1968).
10. Miskus, R. , "DDT, " in G. Zweig, ed. , Analytical Methods for
Pestcides. Plant Growth Regulators, and Food Additives. Vol. 2,
Insecticides, New York, Academic Press, 1964, pp. 97-107.
11. Riemschneider, "No Insecticidal Activity of 1, 1,4, 4-Tetra-
(p-chlorophenyl)-2, 2, 3, 3-Tetrachlorobutane, " J. Amer. Chem.
Soc. , 73, 1374-1375 (1951).
12. Spencer, W. F. , and Cliath, M. M. , "Vapor Density of Dieldrin, "
Environ. Sci. Technol. , 3, 670-674(1969).
85
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13. Spencer, W. F. , and Cliath, M. M. , "Vapor Density and
Apparent Vapor Pressure of LAndane (T -BHC) A. Agr. Food Chem..
18, 529-530 (1970).
14. Spencer, W. F. , Cliath, M. M. , and Farmer, W. J., "Vapor
Density of Soil-Applied Dieldrin as Related to Soil-Water Content,
Temperature, and Dieldrin Concentration, " Soil Sci. Soc.
Amer. Proc. . 33. 509-511 (1969).
15. Hartley, G. S. , "Evaporation of Pesticides, " Advances in
Chemistry JJ6_, Pesticidal Formulations Research, Physical and
Colloidal Chemical Aspects, Amer. Chem. Soc. , Washington,
1969, pp. 115-134.
16. Burchfield, H. P. , and Johnson, D. E. , Guide to the Analysis
of Pesticide Residues, U.S. Department HEW, 1965, Vol. II,
p. VII-D - (DDA).
17. Grummitt, O., Buck, A. C. , and Becker, E. I., "1,1-Di-
(p-chlorophenyl)-ethane, " J. Amer. Chem. Soc. , 67, 2265-2266
(1945).
18. Garbisch, E. W. , Jr. , "Preparation of Some Arylalkenes, "
J. Org. Chem., 26, 4165-4166 (1961).
19. Huntress, E. H., Organic Chlorine Compounds. New York, Wiley,
1948, pp. 383-385.
20. Haller, H. L., et al. , "The Chemical Composition of Technical
DDT," J. Amer. Chem. Soc. . 67, 1591-1602 (1945).
21. Becke, F., and Bucks chewski, H. , 1, l-Bis(p-chlorophenyl)
ethane and 1, l-bis(p-bromophenyl)ethane, Ger 1,269, 116; CA 69.
86989 p. (1969).
22. Bernimolin, J., "Insecticidal Activity of 1, 1, 4, 4-Tetra(p-chloro-
phenyl)-2, 2, 3, 3-Tetrachlorobutane, " J. Amer. Chem. Soc., 71,
2274-2275 (1949).
23. Mount, D. I. , and Brungs, W. A. , "A Simplified Dosing Apparatus
for Fish Toxicology Studies, " Water Research, _1, 21-29 (1967).
24. Mount, D. L , and Stephan, E. C. , "A Method for Establishing
Acceptable Toxicant Limits for Fish - Malathion and the Butoxy-
ethanol Ester of 2, 4-D. " Trans. Am. Fisheries Soc., 9_6,
185-193 (1967).
25. American Public Health Association, Standard Methods for the
Examination of Water and Wastewater, 13th Edition (New York,
1971).
86
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26. Springer, P. F. , "Effects of Herbicides and Fungicides on
Wildlife, " North Carolina Pesticide Manual, North Carolina
State College, Raleigh, N. C. , 1957, pp. 87-106.
27. Henderson, C., Pickering, Q. H., and Tarzwell, C. M.,
"Relative Toxicity of Ten Chlorinated Hydrocarbon Insecticides
to Four Species of Fish, " Trans. Am. Fisheries Soc. , 88,
23-32 (1959). ~~
28. McKee, J. E. , and Wolf, H. W. ed. Water Quality Criteria,
The Resources Agency of California, State Water Quality Control
Board, Sacramento, Calif. 2nd ed. 1963.
29. Anderson, B. G., "The Toxicity of DDT to Daphnia, " Science, 102,
539 (1945).
30. Frear, D. E. H. , and Boyd, J. E. , "Use of Daphnia magna for
the Microbioassay of Pesticides. I. Development of Standardized
Techniques for Rearing Daphnia and Preparation of Dosage-
Mortality Curves for Pesticides, " J. EC on. Entomol. , 60,
1228-1236 (1967).
31. Water Quality Criteria, Federal Water Pollution Control Admin. ,
Washington, D. C. , 1968.
32. Mount, D. I., "Chronic Toxicity of Copper to Fathead Minnows
(Pimephales promelas, Rafinesque). Water Res., 2, 215-223
(1968).
33. Markus, H. J. , "Life History of the Blackhead Minnow (Pimephales
promelas). Copeia, 1934. 116-122.
34. Allison, D. , Kallman, B. J. , Cope, O. B. , and VanValin, C. C. ,
"Insecticides: Effects on Cutthroat Trout of Repeated Exposure
to DDT, " Science, 142. 958-961 (1963).
35. Casey, E. O., and Webb, W. E., "Water Quality Investigations, "
State of Idaho, Dept. of Fish and Game, Boise, Idaho. Project
F-34-R-2, I960.
87
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SECTION X
PATENTS AND PUBLICATIONS
Patent applications will be prepared for significant findings if not
covered by the applications filed under the preceding program,
Contract 14-12-596. Technical papers describing findings on the
chemistry of DDT and toxic testing of products are planned.
89
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SECTION XI
GLOSSARY
CHEMICAL FORMULAS OF PESTICIDES AND DEGRADATION PRODUCTS
DDT* Cl"(Q)"Cr"(Q)"C|
CI-GC I
Cl
2, 2 bis(p-chlorophenyl)-!, 1, 1-trichloroethane
DDD* (TDE) C|
ci-p-ci
H
2,2 bis(p-chlorophenyl}- 1, 1-dichloroethane
DDMS*
CI-C-H
H
2, 2 bis(p-chlorophenyl)- 1-chloroethane
DDEt Cl
H-C-H
H
1, 1 bis(p-chlorophenyl) ethane
DDE
GI'C-CI
2,2 bis(p-chlorophenyl)-1, 1-dichloroethylene
DDMU* (TDEE) Cl -\Cj/C
Cj-C-H
2, 2-bis(p-chlorophenyl)- l~chloroethylene
DDNU*
H-C-H
1, l-bis(p-chlorophenyl) ethylene
* Coding of compounds used by Menzies, "Metabolism
of Pesticides" (Reference 3}
91
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GLOSSARY
CHEMICAL FORMULAS OF PESTICIDES AND DEGRADATION PRODUCTS
DDA
Cl
cr OH
bis(p-chlorophenyl)acetic acid
DBP
Cl
4, 4'-dichlorobenzophenone
DBH
Cl
H
Q
c
I
H
4, 4l-dichlorobenzhydrol
DMC
H
CI9
H
1-hydroxy-1, l-bis(p-chlorophenyl)ethane
TTTB
H-C C — C— C-H
1, 1, 4, 4-tetra(p-chlorophenyl)-2, 2, 3, 3-tetrachlorobutane
TTDB
1, 1, 4, 4-tetra(p-chlorophenyl)-2, 3-dichlorobutene-2
92
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DEFINITION OF TOXICOLOGICAL TERMS
96-hr TLrn - The concentration, estimated from data obtained
via acute toxicity experiments, lethal to 50
percent of the test species during a 96-hr
exposure period.
MATC - The maximum acceptable toxicant concentration.
A term proposed by Mount and Stephan (Reference
24) to denote the toxicant concentration having
no adverse effect upon any stage in the life cycle
of the test species.
Application factor - A constant obtained by dividing the MATC
determined empirically for a given test species
by the 96-hr TLm determined for the same species.
93
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SECTION XII
APPENDIX
Test (Evansville, Indiana) Photoperiod
For Fathead Minnow Chronic Testing
Dawn to Dusk
Time _ Date Day Length (hour and minute)
6:00 - 4:45) Dec. 1 10:45)
6:00 - 4:30) 15 10:30)
6:00 - 4:30) Jan. 1 10:30)
6:00 - 4:45) 15 10:45)
6:00 - 5:15) Feb. 1 11:15) 5-month pre-spawning
6:00 - 5:45) 15 11:45) growth period
6:00 - 6:15) Mar. 1 12:15)
6:00 - 7:00) 15 15 13:00)
6:00 - 7:00) Apr. 1 13:30)
6:00 - 8:15) 15 14:15)
6:00 - 8:45) May 1 14:45)
6:00 - 9:15) 15 15:15)
6:00 - 9:30) June 1 15:30) 4-month spawning
6:00 - 9:45) 15 15:45) period
6:00 - 9:45) July 1 15:45)
6:00 - 9:30) 15 15:30)
I
6:00 - 9:00) Aug. 1 15:00)
6:00 - 8:30 15 14:30)
6:00 - 8:00) Sep. 1 14:00)
6:00 - 7:30) 15 13:30)
Post spacing period
6:00 - 5:30) Nov. 1 11:30)
6:00 - 5:00) 15 11:00)
95
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1 Accession Number
r\ Subject Field & Group
050
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Envirogenics Systems Company
A Division of Aerojet-General Corporation
El Monte, California
Title
DEVELOPMENT OF FIELD-APPLIED DDT
\Avthor(s)
Sweeny, Keith H.
Fischer, James R.
Graefe, Allen F.
Liu, David H.
Marcus, Henry J.
1 .£ Project Designation
EPA Contract
2 ] Note
14-12-922
Citation
Environmental Protection Agency report number, EPA-66o/2-T1*-036, May
Descriptors (Starred First)
* Pesticide Removal, *DDT, ^Reduction (Chemical), *Pesticide Toxicity,
Chlorinated Hydrocarbon Pesticides
Identifiers (Starred First)
# Pesticide Degradation
Abstract Laboratory studies were carried out as a part of initial development of a
concept of controlled destruction of field applied DDT pesticide. Copper catalyzed
ninum reductant was shown to degrade DDT in 24 hr at 25 C and 4 hr at 40 C without
ning DDE. Copper catalyzed iron required a week to reduce DDT at 25 C and 8 hr at
Acidity for field degradation of DDT can be supplied by solid acids such as
amic, oxalic, or citric. An integrated degradable particle was demonstrated by a
reductant particle overlaid with sulfamic acid and coated with DDT. Only moisture
eeded to initiate decomposition. In a demonstration 98. 4% of the DDT was destroyed
days and 99. 8% in 2 weeks at 25°C. Product TTTB is 50-fold less fat soluble than
f and nearly insoluble in water. Product DDEt is 20-fold more soluble than DDT in
er. The vapor pressure of DDEt is about 80-fold greater than DDT. Exposure of
ead minnows, bluegills and rainbow trout to water saturated with DDEt (. 05 ppm) or
TB (. 001 ppm) produced no acute toxic effects. The TLm ofDDEt to Daphnia is about
>pb. Long term chronic exposure of fathead minnows to DDEt saturated waters
wed no effect on adult growth and survival, egg production or hatchability. Growth
survival of freshly hatched fry were affected by DDEt above about . 006 ppm. No
ct on fathead minnow adult or fry growth and survival, egg production or hatchability
shown by TTTB saturated water. Nearly mature fathead minnows consumed 10 mg/kg
weight/day of DDEt or 980 mg/kg body weight/day of TTTB in food without apparent
terious effect. (Sweeny - Envirogenics)
ac/or Keith H. Sweeny institution Envirogenics Systems Company, El Monte, California
(REV. JULY 1B08)
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 2O240
* CPO: 1989-359-339
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